Isolated human G-protein coupled receptors, nucleic acid molecules encoding human GPCR proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the Human genome, the GPCR peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the GPCR peptides and methods of identifying modulators of the GPCR peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of G-Protein coupled receptors (GPCRs) that are related to the leucine-rich repeat-containing GPCR subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel GPCR peptides and proteins and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods. Two alternatively spliced variants are provided, referred to herein as “splice form 1” and “splice form 2”.

BACKGROUND OF THE INVENTION

[0002] G-protein Coupled Receptors

[0003] G-protein coupled receptors (GPCRs) constitute a major class of proteins responsible for transducing a signal within a cell. GPCRs have three structural domains: an amino terminal extracellular domain, a transmembrane domain containing seven transmembrane segments, three extracellular loops, and three intracellular loops, and a carboxy terminal intracellular domain. Upon binding of a ligand to an extracellular portion of a GPCR, a signal is transduced within the cell that results in a change in a biological or physiological property of the cell. GPCRs, along with G-proteins and effectors (intracellular enzymes and channels modulated by G-proteins), are the components of a modular signaling system that connects the state of intracellular second messengers to extracellular inputs.

[0004] GPCR genes and gene-products are potential causative agents of disease (Spiegel et al., J. Clin. Invest. 92:1119-1125 (1993); McKusick et al., J. Med. Genet. 30:1-26 (1993)). Specific defects in the rhodopsin gene and the V2 vasopressin receptor gene have been shown to cause various forms of retinitis pigmentosum (Nathans et al., Annu. Rev. Genet. 26:403-424(1992)), and nephrogenic diabetes insipidus (Holtzman et al., Hum. Mol. Genet. 2:1201-1204 (1993)). These receptors are of critical importance to both the central nervous system and peripheral physiological processes. Evolutionary analyses suggest that the ancestor of these proteins originally developed in concert with complex body plans and nervous systems.

[0005] The GPCR protein superfamily can be divided into five families: Family I, receptors typified by rhodopsin and the β2-purinergic receptor and currently represented by over 200 unique members (Dohlman et al., Annu. Rev. Biochem. 60:653-688 (1991)); Family II, the parathyroid hormone/calcitonin/secretin receptor family (Juppner et al., Science 254:1024-1026 (1991); Lin et al., Science 254:1022-1024 (1991)); Family III, the metabotropic glutamate receptor family (Nakanishi, Science 258 597:603 (1992)); Family IV, the cAMP receptor family, important in the chemotaxis and development of D. discoideum (Klein et al., Science 241:1467-1472 (1988)); and Family V, the fungal mating pheromone receptors such as STE2 (Kurjan, Annu. Rev. Biochem. 61:1097-1129 (1992)).

[0006] There are also a small number of other proteins that present seven putative hydrophobic segments and appear to be unrelated to GPCRs; they have not been shown to couple to G-proteins. Drosophila expresses a photoreceptor-specific protein, bride of sevenless (boss), a seven-transmembrane-segment protein that has been extensively studied and does not show evidence of being a GPCR (Hart et al., Proc. Natl. Acad. Sci. USA 90:5047-5051 (1993)). The gene frizzled (fz) in Drosophila is also thought to be a protein with seven transmembrane segments. Like boss, fz has not been shown to couple to G-proteins (Vinson et al., Nature 338:263-264 (1989)).

[0007] G proteins represent a family of heterotrimeric proteins composed of α, β and γ subunits, that bind guanine nucleotides. These proteins are usually linked to cell surface receptors, e.g., receptors containing seven transmembrane segments. Following ligand binding to the GPCR, a conformational change is transmitted to the G protein, which causes the α-subunit to exchange a bound GDP molecule for a GTP molecule and to dissociate from the βγ-subunits. The GTP-bound form of the α-subunit typically functions as an effector-modulating moiety, leading to the production of second messengers, such as cAMP (e.g., by activation of adenyl cyclase), diacylglycerol or inositol phosphates. Greater than 20 different types of α-subunits are known in humans. These subunits associate with a smaller pool of β and γ subunits. Examples of mammalian G proteins include Gi, Go, Gq, Gs and Gt. G proteins are described extensively in Lodish et al., Molecular Cell Biology, (Scientific American Books Inc., New York, N.Y., 1995), the contents of which are incorporated herein by reference. GPCRs, G proteins and G protein-linked effector and second messenger systems have been reviewed in The G-Protein Linked Receptor Fact Book, Watson et al., eds., Academic Press (1994).

[0008] Aminergic GPCRs

[0009] One family of the GPCRS, Family II, contains receptors for acetylcholine, catecholamine, and indoleamine ligands (hereafter referred to as biogenic amines). The biogenic amine receptors (aminergic GPCRs) represent a large group of GPCRs that share a common evolutionary ancestor and which are present in both vertebrate (deuterostome), and invertebrate (protostome) lineages. This family of GPCRs includes, but is not limited to the 5-HT-like, the dopamine-like, the acetylcholine-like, the adrenaline-like and the melatonin-like GPCRs.

[0010] Dopamine Receptors

[0011] The understanding of the dopaminergic system relevance in brain function and disease developed several decades ago from three diverse observations following drug treatments. These were the observations that dopamine replacement therapy improved Parkinson's disease symptoms, depletion of dopamine and other catecholamines by reserpine caused depression and antipsychotic drugs blocked dopamine receptors. The finding that the dopamine receptor binding affinities of typical antipsychotic drugs correlate with their clinical potency led to the dopamine overactivity hypothesis of schizophrenia (Snyder, S. H., Am J Psychiatry 133, 197-202 (1976); Seeman, P. and Lee, T., Science 188, 1217-9 (1975)). Today, dopamine receptors are crucial targets in the pharmacological therapy of schizophrenia, Parkinson's disease, Tourette's syndrome, tardive dyskinesia and Huntington's disease. The dopaminergic system includes the nigrostriatal, mesocorticolimbic and tuberoinfundibular pathways. The nigrostriatal pathway is part of the striatal motor system and its degeneration leads to Parkinson's disease; the mesocorticolimbic pathway plays a key role in reinforcement and in emotional expression and is the desired site of action of antipsychotic drugs; the tuberoinfundibular pathways regulates prolactin secretion from the pituitary.

[0012] Dopamine receptors are members of the G protein coupled receptor superfamily, a large group proteins that share a seven helical membrane-spanning structure and transduce signals through coupling to heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins). Dopamine receptors are classified into subfamilies: D1-like (D1 and D5) and D2-like (D2, D3 and D4) based on their different ligand binding profiles, signal transduction properties, sequence homologies and genomic organizations (Civelli, O., Bunzow, J. R. and Grandy, D. K., Annu Rev Pharmacol Toxicol 33, 281-307 (1993)). The D1-like receptors, D1 and D5, stimulate cAMP synthesis through coupling with Gs-like proteins and their genes do not contain introns within their protein coding regions. On the other hand, the D2-like receptors, D2, D3 and D4, inhibit cAMP synthesis through their interaction with Gi-like proteins and share a similar genomic organization which includes introns within their protein coding regions.

[0013] Serotonin Receptors

[0014] Serotonin (5-Hydroxytryptamine; 5-HT) was first isolated from blood serum, where it was shown to promote vasoconstriction (Rapport, M. M., Green, A. A. and Page, I. H., J Biol Chem 176, 1243-1251 (1948). Interest on a possible relationship between 5-HT and psychiatric disease was spurred by the observations that hallucinogens such as LSD and psilocybin inhibit the actions of 5-HT on smooth muscle preparations (Gaddum, J. H. and Hameed, K. A., Br J Pharmacol 9, 240-248 (1954)). This observation lead to the hypothesis that brain 5-HT activity might be altered in psychiatric disorders (Wooley, D. W. and Shaw, E., Proc Natl Acad Sci USA 40, 228-231 (1954); Gaddum, J. H. and Picarelli, Z. P., Br J Pharmacol 12, 323-328 (1957)). This hypothesis was strengthened by the introduction of tricyclic antidepressants and monoamine oxidase inhibitors for the treatment of major depression and the observation that those drugs affected noradrenaline and 5-HT metabolism. Today, drugs acting on the serotoninergic system have been proved to be effective in the pharmacotherapy of psychiatric diseases such as depression, schizophrenia, obsessive-compulsive disorder, panic disorder, generalized anxiety disorder and social phobia as well as migraine, vomiting induced by cancer chemotherapy and gastric motility disorders.

[0015] Serotonin receptors represent a very large and diverse family of neurotransmitter receptors. To date thirteen 5-HT receptor proteins coupled to G proteins plus one ligand-gated ion channel receptor (5-HT3) have been described in mammals. This receptor diversity is thought to reflect serotonin's ancient origin as a neurotransmitter and a hormone as well as the many different roles of 5-HT in mammals. The 5-HT receptors have been classified into seven subfamilies or groups according to their different ligand-binding affinity profiles, molecular structure and intracellular transduction mechanisms (Hoyer, D. et al., Pharmacol. Rev. 46, 157-203 (1994)).

[0016] Adrenergic GPCRs

[0017] The adrenergic receptors comprise one of the largest and most extensively characterized families within the G-protein coupled receptor “superfamily”. This superfamily includes not only adrenergic receptors, but also muscarinic, cholinergic, dopaminergic, serotonergic, and histaminergic receptors. Numerous peptide receptors include glucagon, somatostatin, and vasopressin receptors, as well as sensory receptors for vision (rhodopsin), taste, and olfaction, also belong to this growing family. Despite the diversity of signalling molecules, G-protein coupled receptors all possess a similar overall primary structure, characterized by 7 putative membrane-spanning .alpha. helices (Probst et al., 1992). In the most basic sense, the adrenergic receptors are the physiological sites of action of the catecholamines, epinephrine and norepinephrine. Adrenergic receptors were initially classified as either .alpha. or .beta. by Ahlquist, who demonstrated that the order of potency for a series of agonists to evoke a physiological response was distinctly different at the 2 receptor subtypes (Ahlquist, 1948). Functionally, .alpha. adrenergic receptors were shown to control vasoconstriction, pupil dilation and uterine inhibition, while .beta. adrenergic receptors were implicated in vasorelaxation, myocardial stimulation and bronchodilation (Regan et al., 1990). Eventually, pharmacologists realized that these responses resulted from activation of several distinct adrenergic receptor subtypes. .beta. adrenergic receptors in the heart were defined as .beta..sub.1, while those in the lung and vasculature were termed .beta..sub.2 (Lands et al., 1967).

[0018] .alpha. Adrenergic receptors, meanwhile, were first classified based on their anatomical location, as either pre or post-synaptic (.alpha..sub.2 and .alpha..sub.1, respectively) (Langer et al., 1974). This classification scheme was confounded, however, by the presence of .alpha..sub.2 receptors in distinctly non-synaptic locations, such as platelets (Berthelsen and Pettinger, 1977). With the development of radioligand binding techniques, .alpha. adrenergic receptors could be distinguished pharmacologically based on their affinities for the antagonists prazosin or yohimbine (Stark, 1981). Definitive evidence for adrenergic receptor subtypes, however, awaited purification and molecular cloning of adrenergic receptor subtypes. In 1986, the genes for the hamster .beta..sub.2 (Dickson et al., 1986) and turkey .beta..sub.1 adrenergic receptors (Yarden et al., 1986) were cloned and sequenced. Hydropathy analysis revealed that these proteins contain 7 hydrophobic domains similar to rhodopsin, the receptor for light. Since that time the adrenergic receptor family has expanded to include 3 subtypes of .beta. receptors (Emorine et al., 1989), 3 subtypes of .alpha..sub.1 receptors (Schwinn et al., 1990), and 3 distinct types of .beta..sub.2 receptors (Lomasney et al., 1990).

[0019] The cloning, sequencing and expression of alpha receptor subtypes from animal tissues has led to the subclassification of the alpha 1 receptors into alpha 1d (formerly known as alpha 1a or 1a/1d), alpha 1b and alpha 1a (formerly known as alpha 1c) subtypes. Each alpha 1 receptor subtype exhibits its own pharmacologic and tissue specificities. The designation “alpha 1a” is the appellation recently approved by the IUPHAR Nomenclature Committee for the previously designated “alpha 1c” cloned subtype as outlined in the 1995 Receptor and Ion Channel Nomenclature Supplement (Watson and Girdlestone, 1995). The designation alpha 1a is used throughout this application to refer to this subtype. At the same time, the receptor formerly designated alpha 1a was renamed alpha 1d. The new nomenclature is used throughout this application. Stable cell lines expressing these alpha 1 receptor subtypes are referred to herein; however, these cell lines were deposited with the American Type Culture Collection (ATCC) under the old nomenclature. For a review of the classification of alpha 1 adrenoceptor subtypes, see, Martin C. Michel, et al., Naunyn-Schmiedeberg's Arch. Pharmacol. (1995) 352:1-10.

[0020] The differences in the alpha adrenergic receptor subtypes have relevance in pathophysiologic conditions. Benign prostatic hyperplasia, also known as benign prostatic hypertrophy or BPH, is an illness typically affecting men over fifty years of age, increasing in severity with increasing age. The symptoms of the condition include, but are not limited to, increased difficulty in urination and sexual dysfunction. These symptoms are induced by enlargement, or hyperplasia, of the prostate gland. As the prostate increases in size, it impinges on free-flow of fluids through the male urethra. Concommitantly, the increased noradrenergic innervation of the enlarged prostate leads to an increased adrenergic tone of the bladder neck and urethra, further restricting the flow of urine through the urethra.

[0021] The .alpha..sub.2 receptors appear to have diverged rather early from either .beta. or .alpha..sub.1 receptors. The .alpha..sub.2 receptors have been broken down into 3 molecularly distinct subtypes termed .alpha..sub.2 C2, .alpha..sub.2 C4, and .alpha..sub.2 C10 based on their chromosomal location. These subtypes appear to correspond to the pharmacologically defined .alpha..sub.2B, .alpha..sub.2C, and .alpha..sub.2A subtypes, respectively (Bylund et al., 1992). While all the receptors of the adrenergic type are recognized by epinephrine, they are pharmacologically distinct and are encoded by separate genes. These receptors are generally coupled to different second messenger pathways that are linked through G-proteins. Among the adrenergic receptors, .beta..sub.1 and .beta..sub.2 receptors activate the adenylate cyclase, .alpha..sub.2 receptors inhibit adenylate cyclase and .alpha..sub.1 receptors activate phospholipase C pathways, stimulating breakdown of polyphosphoinositides (Chung, F. Z. et al., J. Biol. Chem., 263:4052 (1988)). .alpha..sub.1 and .alpha..sub.2 adrenergic receptors differ in their cell activity for drugs.

[0022] Issued U.S. patent that disclose the utility of members of this family of proteins include, but are not limited to, U.S. Pat. No. 6,063,785 Phthalimido arylpiperazines useful in the treatment of benign prostatic hyperplasia; U.S. Pat. No. 6,060,492 Selective .beta.3 adrenergic agonists; U.S. Pat. No. 6,057,350 Alpha 1a adrenergic receptor antagonists; U.S. Pat. No. 6,046,192 Phenylethanolaminotetralincarboxamide derivatives; U.S. Pat. No. 6,046,183 Method of synergistic treatment for benign prostatic hyperplasia; U.S. Pat. No. 6,043,253 Fused piperidine substituted arylsulfonamides as .beta.3-agonists; U.S. Pat. No. 6,043,224 Compositions and methods for treatment of neurological disorders and neurodegenerative diseases; U.S. Pat. No. 6,037,354 Alpha 1a adrenergic receptor antagonists; U.S. Pat. No. 6,034,106 Oxadiazole benzenesulfonamides as selective .beta..sub.3 Agonist for the treatment of Diabetes and Obesity; U.S. Pat. No. 6,011,048 Thiazole benzenesulfonamides as .beta.3 agonists for treatment of diabetes and obesity; U.S. Pat. No. 6,008,361 5,994,506 Adrenergic receptor; U.S. Pat. No. 5,994,294 Nitrosated and nitrosylated .alpha.-adrenergic receptor antagonist compounds, compositions and their uses; U.S. Pat. No. 5,990,128 .alpha..sub.1C specific compounds to treat benign prostatic hyperplasia; U.S. Pat. No. 5,977,154 Selective .beta.3 adrenergic agonist; U.S. Pat. No. 5,977,115 Alpha 1a adrenergic receptor antagonists; U.S. Pat. No. 5,939,443 Selective .beta.3 adrenergic agonists; U.S. Pat. No. 5,932,538 Nitrosated and nitrosylated .alpha.-adrenergic receptor antagonist compounds, compositions and their uses; U.S. Pat. No. 5,922,722 Alpha 1a adrenergic receptor antagonists 26 U.S. Pat. Nos. 5,908,830 and 5,861,309 DNA endoding human alpha 1 adrenergic receptors.

[0023] Purinergic GPCRs

[0024] Purinoceptor P2Y1

[0025] P2 purinoceptors have been broadly classified as P2X receptors which are ATP-gated channels; P2Y receptors, a family of G protein-coupled receptors, and P2Z receptors, which mediate nonselective pores, in mast cells. Numerous subtypes have been identified for each of the P2 receptor classes. P2Y receptors are characterized by their selective responsiveness towards ATP and its analogs. Some respond also to UTP. Based on the recommendation for nomenclature of P2 purinoceptors, the P2Y purinoceptors were numbered in the order of cloning. P2Y1, P2Y2 and P2Y3 have been cloned from a variety of species. P2Y1 responds to both ADP and ATP. Analysis of P2Y receptor subtype expression in human bone and 2 osteoblastic cell lines by RT-PCR showed that all known human P2Y receptor subtypes were expressed: P2Y1, P2Y2, P2Y4, P2Y6, and P2Y7 (Maier et al. 1997). In contrast, analysis of brain-derived cell lines suggested that a selective expression of P2Y receptor subtypes occurs in brain tissue.

[0026] Leon et al. generated P2Y1-null mice to define the physiologic role of the P2Y1 receptor. (J. Clin. Invest. 104: 1731-1737(1999)) These mice were viable with no apparent abnormalities affecting their development, survival, reproduction, or morphology of platelets, and the platelet count in these animals was identical to that of wildtype mice. However, platelets from P2Y1-deficient mice were unable to aggregate in response to usual concentrations of ADP and displayed impaired aggregation to other agonists, while high concentrations of ADP induced platelet aggregation without shape change. In addition, ADP-induced inhibition of adenylyl cyclase still occurred, demonstrating the existence of an ADP receptor distinct from P2Y1. P2Y1-null mice had no spontaneous bleeding tendency but were resistant to thromboembolism induced by intravenous injection of ADP or collagen and adrenaline. Hence, the P2Y1 receptor plays an essential role in thrombotic states and represents a potential target for antithrombotic drugs. Somers et al. mapped the P2RY1 gene between flanking markers D3S1279 and D3S1280 at a position 173 to 174 cM from the most telomeric markers on the short arm of chromosome 3. (Genomics 44: 127-130 (1997)).

[0027] Purinoceptor P2Y2

[0028] The chloride ion secretory pathway that is defective in cystic fibrosis (CF) can be bypassed by an alternative pathway for chloride ion transport that is activated by extracellular nucleotides. Accordingly, the P2 receptor that mediates this effect is a therapeutic target for improving chloride secretion in CF patients. Parr et al. reported the sequence and functional expression of a cDNA cloned from human airway epithelial cells that encodes a protein with properties of a P2Y nucleotide receptor. (Proc. Nat. Acad. Sci. 91: 3275-3279 (1994)) The human P2RY2 gene was mapped to chromosome 11q13.5-q14.1.

[0029] Purinoceptor P2RY4

[0030] The P2RY4 receptor appears to be activated specifically by UTP and UDP, but not by ATP and ADP. Activation of this uridine nucleotide receptor resulted in increased inositol phosphate formation and calcium mobilization. The UNR gene is located on chromosome Xq13.

[0031] Purinoceptor P2Y6

[0032] Somers et al. mapped the P2RY6 gene to 11q13.5, between polymorphic markers D11S1314 and D11S916, and P2RY2 maps within less than 4 cM of P2RY6. (Genomics 44: 127-130 (1997)) This was the first chromosomal clustering of this gene family to be described.

[0033] Adenine and uridine nucleotides, in addition to their well established role in intracellular energy metabolism, phosphorylation, and nucleic acid synthesis, also are important extracellular signaling molecules. P2Y metabotropic receptors are GPCRs that mediate the effects of extracellular nucleotides to regulate a wide variety of physiological processes. At least ten subfamilies of P2Y receptors have been identified. These receptor subfamilies differ greatly in their sequences and in their nucleotide agonist selectivities and efficacies.

[0034] It has been demonstrated that the P2Y1 receptors are strongly expressed in the brain, but the P2Y2, P2Y4 and P2Y6 receptors are also present. The localisation of one or more of these subtypes on neurons, on glia cells, on brain vasculature or on ventricle ependimal cells was found by in situ mRNA hybridisation and studies on those cells in culture. The P2Y1 receptors are prominent on neurons. The coupling of certain P2Y receptor subtypes to N-type Ca2+ channels or to particular K+ channels was also demonstrated.

[0035] It has also been demonstrated that several P2Y receptors mediate potent growth stimulatory effects on smooth muscle cells by stimulating intracellular pathways including Gq-proteins, protein kinase C and tyrosine phosphorylation, leading to increased immediate early gene expression, cell number, DNA and protein synthesis. It has been further demonstrated that P2Y regulation plays a mitogenic role in response to the development of artherosclerosis.

[0036] It has further been demonstrated that P2Y receptors play a critical role in cystic fibrosis. The volume and composition of the liquid that lines the airway surface is modulated by active transport of ions across the airway epithelium. This in turn is regulated both by autonomic agonists acting on basolateral receptors and by agonists acting on luminal receptors. Specifically, extracellular nucleotides present in the airway surface liquid act on luminal P2Y receptors to control both Cl− secretion and Na+ absorption. Since nucleotides are released in a regulated manner from airway epithelial cells, it is likely that their control over airway ion transport forms part of an autocrine regulatory system localised to the luminal surface of airway epithelia. In addition to this physiological role, P2Y receptor agonists have the potential to be of crucial benefit in the treatment of CF, a disorder of epithelial ion transport. The airways of people with CF have defective Cl− secretion and abnormally high rates of Na+ absorption. Since P2Y receptor agonists can regulate both these ion transport pathways they have the potential to pharmacologically bypass the ion transport defects in CF.

[0037] Leucine-rich Repeat-containing GPCRs (LGRs)

[0038] The present invention provides two human alternative protein splice forms (referred to herein as “splice form 1” and “splice form 2”), and the encoding gene, that are related to the family of leucine-rich repeat-containing GPCRs (LGRs). These alternative splice forms show the highest degree of similarity to LGR6 (see the amino acid sequence alignments provided in FIG. 2).

[0039] LGRs are paralogs of glycoprotein hormone receptors such as LH receptor, FSH receptor, and TSH receptor. These glycoprotein hormone receptors are unique among the GPCR family in having large N-terminal extracellular domains that are important for interacting with and binding glycoprotein hormone ligands. LGR7 contains a low-density lipoprotein receptor cysteine-rich motif at the N-terminus and LGR6 has a hinge region after leucine-rich repeats. LGR7 action is thought to be mediated by the protein kinase A pathway rather than the phospholipase C pathway (Hsu et al., Mol Endocrinol August 2000; 14(8):1257-71).

[0040] For a further review of relevant LGRs, see Nishi et al., Endocrinology November 2000; 141(11):4081-90 and Tensen et al., Proc Natl Acad Sci USA May 24, 1994; 91(11):4816-20.

[0041] Due to their importance in endocrine physiology, novel human LGR proteins/genes, such as provided by the present invention, are valuable as potential targets for the development of therapeutics to treat endocrine-related disorders, as well as other disorders. Furthermore, SNPs in LGR genes, such as provided by the present invention, may serve as valuable markers for the diagnosis, prognosis, prevention, and/or treatment of such disorders.

[0042] Using the information provided by the present invention, reagents such as probes/primers for detecting the SNPs or the expression of the protein/gene provided herein may be readily developed and, if desired, incorporated into kit formats such as nucleic acid arrays, primer extension reactions coupled with mass spec detection (for SNP detection), or TaqMan PCR assays (Applied Biosystems, Foster City, Calif.).

[0043] GPCRs, particularly members of the leucine-rich repeat-containing GPCR subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown GPCRs. The present invention advances the state of the art by providing a previously unidentified human GPCR.

SUMMARY OF THE INVENTION

[0044] The present invention is based in part on the identification of nucleic acid sequences that encode amino acid sequences of human GPCR peptides and proteins that are related to the leucine-rich repeat-containing GPCR subfamily, allelic variants thereof and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents. Two alternatively spliced variants are provided, referred to herein as “splice form 1” and “splice form 2”.

[0045] The proteins of the present inventions are GPCRs that participate in signaling pathways mediated by the leucine-rich repeat-containing GPCR subfamily in cells that express these proteins. Experimental data as provided in FIG. 1 indicates that splice forms 1 and 2 are expressed in head/neck tissue, infant brain, and stomach. Splice form 2 is also expressed in colon tumors and leukocytes. As used herein, a “signaling pathway” refers to the modulation (e.g., stimulation or inhibition) of a cellular function/activity upon the binding of a ligand to the GPCR protein. Examples of such functions include mobilization of intracellular molecules that participate in a signal transduction pathway, e.g., phosphatidylinositol 4,5-bisphosphate (PIP₂), inositol 1,4,5-triphosphate (IP₃) and adenylate cyclase; polarization of the plasma membrane; production or secretion of molecules; alteration in the structure of a cellular component; cell proliferation, e.g., synthesis of DNA; cell migration; cell differentiation; and cell survival

[0046] The response mediated by the receptor protein depends on the type of cell it is expressed on. Some information regarding the types of cells that express other members of the subfamily of GPCRs of the present invention is already known in the art (see references cited in Background and information regarding closest homologous protein provided in FIG. 2; Experimental data as provided in FIG. 1 indicates that splice forms 1 and 2 are expressed in head/neck tissue, infant brain, and stomach. Splice form 2 is also expressed in colon tumors and leukocytes.). For example, in some cells, binding of a ligand to the receptor protein may stimulate an activity such as release of compounds, gating of a channel, cellular adhesion, migration, differentiation, etc., through phosphatidylinositol or cyclic AMP metabolism and turnover while in other cells, the binding of the ligand will produce a different result. Regardless of the cellular activity/response modulated by the particular GPCR of the present invention, a skilled artisan will clearly know that the receptor protein is a GPCR and interacts with G proteins to produce one or more secondary signals, in a variety of intracellular signal transduction pathways, e.g., through phosphatidylinositol or cyclic AMP metabolism and turnover, in a cell thus participating in a biological process in the cells or tissues that express the GPCR. Experimental data as provided in FIG. 1 indicates that GPCR splice forms 1 and 2 of the present invention are expressed in head/neck tissue, infant brain, and stomach, as indicated by virtual northern blot analysis. Virtual northern blot analysis also indicates that splice form 2 is expressed in colon tumors and, additionally, PCR-based tissue screening panels indicate that splice form 2 is also expressed in leukocytes.

[0047] As used herein, “phosphatidylinositol turnover and metabolism” refers to the molecules involved in the turnover and metabolism of phosphatidylinositol 4,5-bisphosphate (PIP₂) as well as to the activities of these molecules. PIP₂ is a phospholipid found in the cytosolic leaflet of the plasma membrane. Binding of ligand to the receptor activates, in some cells, the plasma-membrane enzyme phospholipase C that in turn can hydrolyze PIP₂ to produce 1,2-diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). Once formed IP₃ can diffuse to the endoplasmic reticulum surface where it can bind an IP₃ receptor, e.g., a calcium channel protein containing an IP₃ binding site. IP_(b 3) binding can induce opening of the channel, allowing calcium ions to be released into the cytoplasm. IP₃ can also be phosphorylated by a specific kinase to form inositol 1,3,4,5-tetraphosphate (IP₄), a molecule that can cause calcium entry into the cytoplasm from the extracellular medium. IP₃ and IP₄ can subsequently be hydrolyzed very rapidly to the inactive products inositol 1,4-biphosphate (IP₂) and inositol 1,3,4-triphosphate, respectively. These inactive products can be recycled by the cell to synthesize PIP₂. The other second messenger produced by the hydrolysis of PIP₂, namely 1,2-diacylglycerol (DAG), remains in the cell membrane where it can serve to activate the enzyme protein kinase C. Protein kinase C is usually found soluble in the cytoplasm of the cell, but upon an increase in the intracellular calcium concentration, this enzyme can move to the plasma membrane where it can be activated by DAG. The activation of protein kinase C in different cells results in various cellular responses such as the phosphorylation of glycogen synthase, or the phosphorylation of various transcription factors, e.g., NF-kB. The language “phosphatidylinositol activity”, as used herein, refers to an activity of PIP₂ or one of its metabolites.

[0048] Another signaling pathway in which the receptor may participate is the cAMP turnover pathway. As used herein, “cyclic AMP turnover and metabolism” refers to the molecules involved in the turnover and metabolism of cyclic AMP (cAMP) as well as to the activities of these molecules. Cyclic AMP is a second messenger produced in response to ligand-induced stimulation of certain G protein coupled receptors. In the cAMP signaling pathway, binding of a ligand to a GPCR can lead to the activation of the enzyme adenyl cyclase, which catalyzes the synthesis of cAMP. The newly synthesized cAMP can in turn activate a cAMP-dependent protein kinase. This activated kinase can phosphorylate a voltage-gated potassium channel protein, or an associated protein, and lead to the inability of the potassium channel to open during an action potential. The inability of the potassium channel to open results in a decrease in the outward flow of potassium, which normally repolarizes the membrane of a neuron, leading to prolonged membrane depolarization.

[0049] By targeting an agent to modulate a GPCR, the signaling activity and biological process mediated by the receptor can be agonized or antagonized in specific cells and tissues. Experimental data as provided in FIG. 1 indicates that splice forms 1 and 2 are expressed in head/neck tissue, infant brain, and stomach. Splice form 2 is also expressed in colon tumors and leukocytes. Such agonism and antagonism serves as a basis for modulating a biological activity in a therapeutic context (mammalian therapy) or toxic context (anti-cell therapy, e.g. anti-cancer agent).

DESCRIPTION OF THE FIGURE SHEETS

[0050]FIG. 1 provides nucleotide sequences of a transcript sequence of splice form 1 (SEQ ID NO:1) and a cDNA molecule for splice form 2 (SEQ ID NO:4). In addition, structure and functional information is provided for each splice form, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of the inventions based on these molecular sequences. Experimental data as provided in FIG. 1 indicates that splice forms 1 and 2 are expressed in head/neck tissue, infant brain, and stomach. Splice form 2 is also expressed in colon tumors and leukocytes.

[0051]FIG. 2 provides the predicted amino acid sequences of GPCR splice form 1 (SEQ ID NO:2) and splice form 2 (SEQ ID NO:5) of the present invention. In addition, structure and functional information such as protein family, function, and modification sites is provided for each splice form where available, allowing one to readily determine specific uses of the inventions based on these molecular sequences. FIG. 2 also provides an amino acid sequence alignment of splice form 1 and splice form 2, indicating the differences in the amino acid sequences of the two splice forms.

[0052]FIG. 3 provides the genomic sequence (SEQ ID NO:3) that spans the gene encoding the GPCR proteins of the present invention. In addition structure and functional information, such as intron/exon structure, promoter location, etc., is provided where available, allowing one to readily determine specific uses of the inventions based on this molecular sequence. As illustrated in FIG. 3, SNPs were identified at 107 different nucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

[0053] General Description

[0054] The present invention is based on the sequencing of the human genome. During the sequencing and assembly of the human genome, analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homology to protein/peptide/domains identified and characterized within the art as being a GPCR protein or part of a GPCR protein, that are related to the leucine-rich repeat-containing GPCR subfamily. Utilizing these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized. Based on this analysis, the present invention provides amino acid sequences of human GPCR peptides and proteins that are related to the leucine-rich repeat-containing GPCR subfamily, nucleic acid sequences in the form of transcript sequences, cDNA sequences and/or genomic sequences that encode these GPCR peptides and proteins, nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known protein/peptide/domain that has structural or sequence homology to the GPCR of the present invention. Two alternatively spliced variants are provided, referred to herein as “splice form 1” and “splice form 2”.

[0055] In addition to being previously unknown, the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology and/or structural relatedness to known GPCR proteins of the leucine-rich repeat-containing GPCR subfamily and the expression pattern observed. Experimental data as provided in FIG. 1 indicates that splice forms 1 and 2 are expressed in head/neck tissue, infant brain, and stomach. Splice form 2 is also expressed in colon tumors and leukocytes. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene. Some of the more specific features of the peptides of the present invention, and the uses thereof, are described herein, particularly in the Background of the Invention and in the annotation provided in the Figures, and/or are known within the art for each of the known leucine-rich repeat-containing GPCR family or subfamily of GPCR proteins.

[0056] Specific Embodiments

[0057] Peptide Molecules

[0058] The present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the GPCR family of proteins and are related to the leucine-rich repeat-containing GPCR subfamily (protein sequences are provided in FIG. 2, transcript/cDNA sequences are provided in FIG. 1 and genomic sequences are provided in FIG. 3). The peptide sequences provided in FIG. 2, as well as the obvious variants described herein, particularly allelic variants as identified herein and using the information in FIG. 3, will be referred herein as the GPCR peptides of the present invention, GPCR peptides, or peptides/proteins of the present invention.

[0059] The present invention provides isolated peptide and protein molecules that consist of, consist essentially of, or comprise the amino acid sequences of the GPCR peptides disclosed in FIG. 2, (encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNA sequence, or FIG. 3, genomic sequence), as well as all obvious variants of these peptides that are within the art to make and use. Some of these variants are described in detail below.

[0060] As used herein, a peptide is said to be “isolated” or “purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below).

[0061] In some uses, “substantially free of cellular material” includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation.

[0062] The language “substantially free of chemical precursors or other chemicals” includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the GPCR peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.

[0063] The isolated GPCR peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. Experimental data as provided in FIG. 1 indicates that splice forms 1 and 2 are expressed in head/neck tissue, infant brain, and stomach. Splice form 2 is also expressed in colon tumors and leukocytes. For example, a nucleic acid molecule encoding the GPCR peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Many of these techniques are described in detail below.

[0064] Accordingly, the present invention provides proteins that consist of the amino acid sequences provided in FIG. 2 (SEQ ID NOS:2 and 5), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NOS:1 and 4) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence of such a protein is provided in FIG. 2. A protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.

[0065] The present invention further provides proteins that consist essentially of the amino acid sequences provided in FIG. 2 (SEQ ID NOS:2 and 5), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NOS:1 and 4) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein.

[0066] The present invention further provides proteins that comprise the amino acid sequences provided in FIG. 2 (SEQ ID NOS:2 and 5), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NOS:1 and 4) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. Ih such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the GPCR peptides of the present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be made/isolated is provided below.

[0067] The GPCR peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins. Such chimeric and fusion proteins comprise a GPCR peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the GPCR peptide. “Operatively linked” indicates that the GPCR peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the GPCR peptide.

[0068] In some uses, the fusion protein does not affect the activity of the GPCR peptide per se. For example, the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant GPCR peptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence.

[0069] A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A GPCR peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the GPCR peptide.

[0070] As mentioned above, the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.

[0071] Such variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the GPCR peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs.

[0072] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0073] The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0074] The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0075] Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the GPCR peptides of the present invention as well as being encoded by the same genetic locus as the GPCR peptide provided herein. The gene encoding the novel GPCR proteins of the present invention is located on a genome component that has been mapped to human chromosome 1 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

[0076] Allelic variants of a GPCR peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the GPCR peptide as well as being encoded by the same genetic locus as the GPCR peptide provided herein. Genetic locus can readily be determined based on the genomic information provided in FIG. 3, such as the genomic sequence mapped to the reference human. The gene encoding the novel GPCR proteins of the present invention is located on a genome component that has been mapped to human chromosome 1 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70-80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a GPCR peptide encoding nucleic acid molecule under stringent conditions as more fully described below.

[0077]FIG. 3 provides information on SNPs that have been found in the gene encoding the GPCR proteins of the present invention. SNPs were identified at 107 different nucleotide positions. Some of these SNPs that are located outside the ORF and in introns may affect gene expression.

[0078] Paralogs of a GPCR peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the GPCR peptide, as being encoded by a gene from humans, and as having similar activity or function. Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a GPCR peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.

[0079] Orthologs of a GPCR peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the GPCR peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a GPCR peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins.

[0080] Non-naturally occurring variants of the GPCR peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the GPCR peptide. For example, one class of substitutions are conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a GPCR peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

[0081] Variant GPCR peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind ligand, ability to bind G-protein, ability to mediate signaling, etc. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. FIG. 2 provides the result of protein analysis that identifies critical domains/regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.

[0082] Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

[0083] Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085 (1989)), particularly using the results provided in FIG. 2. The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as ligand/effector molecule binding or in assays such as an in vitro proliferative activity. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

[0084] The present invention further provides fragments of the GPCR peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in FIG. 2. The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention.

[0085] As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from a GPCR peptide. Such fragments can be chosen based on the ability to retain one or more of the biological activities of the GPCR peptide or could be chosen for the ability to perform a function, e.g. ability to bind ligand or effector molecule or act as an immunogen. Particularly important fragments are biologically active fragments, peptides which are, for example, about 8 or more amino acids in length. Such fragments will typically comprise a domain or motif of the GPCR peptide, e.g., active site, a G-protein binding site, a transmembrane domain or a ligand-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well-known and readily available to those of skill in the art (e.g., PROSITE analysis). The results of one such analysis are provided in FIG. 2.

[0086] Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in GPCR peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art(some of these features are identified in FIG. 2).

[0087] Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0088] Such modifications are well-known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62 (1992)).

[0089] Accordingly, the GPCR peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature GPCR peptide is fused with another compound, such as a compound to increase the half-life of the GPCR peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature GPCR peptide, such as a leader or secretory sequence or a sequence for purification of the mature GPCR peptide or a pro-protein sequence.

[0090] Protein/Peptide Uses

[0091] The proteins of the present invention can be used in substantial and specific assays related to the functional information provided in the Figures and Back Ground Section; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or receptor) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the protein can be used to identify the binding partner so as to develop a system to identify inhibitors of the binding interaction. Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as commercial products.

[0092] Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0093] The potential uses of the peptides of the present invention are based primarily on the source of the protein as well as the class/action of the protein. For example, GPCRs isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the GPCR. Experimental data as provided in FIG. 1 indicates that GPCR splice forms 1 and 2 of the present invention are expressed in head/neck tissue, infant brain, and stomach, as indicated by virtual northern blot analysis. Virtual northern blot analysis also indicates that splice form 2 is expressed in colon tumors and, additionally, PCR-based tissue screening panels indicate that splice form 2 is also expressed in leukocytes. Approximately 70% of all pharmaceutical agents modulate the activity of a GPCR. A combination of the invertebrate and mammalian ortholog can be used in selective screening methods to find agents specific for invertebrates. The structural and functional information provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention, particularly in combination with the expression information provided in FIG. 1. Experimental data as provided in FIG. 1 indicates that splice forms 1 and 2 are expressed in head/neck tissue, infant brain, and stomach. Splice form 2 is also expressed in colon tumors and leukocytes. Such uses can readily be determined using the information provided herein, that known in the art and routine experimentation.

[0094] The proteins of the present invention (including variants and fragments that may have been disclosed prior to the present invention) are useful for biological assays related to GPCRs that are related to members of the leucine-rich repeat-containing GPCR subfamily. Such assays involve any of the known GPCR functions or activities or properties useful for diagnosis and treatment of GPCR-related conditions that are specific for the subfamily of GPCRs that the one of the present invention belongs to, particularly in cells and tissues that express this receptor. Experimental data as provided in FIG. 1 indicates that GPCR splice forms 1 and 2 of the present invention are expressed in head/neck tissue, infant brain, and stomach, as indicated by virtual northern blot analysis. Virtual northern blot analysis also indicates that splice form 2 is expressed in colon tumors and, additionally, PCR-based tissue screening panels indicate that splice form 2 is also expressed in leukocytes.

[0095] The proteins of the present invention are also useful in drug screening assays, in cell-based or cell-free systems. Cell-based systems can be native, i.e., cells that normally express the receptor protein, as a biopsy or expanded in cell culture. Experimental data as provided in FIG. 1 indicates that splice forms 1 and 2 are expressed in head/neck tissue, infant brain, and stomach. Splice form 2 is also expressed in colon tumors and leukocytes. In an alternate embodiment, cell-based assays involve recombinant host cells expressing the receptor protein.

[0096] The polypeptides can be used to identify compounds that modulate receptor activity of the protein in its natural state, or an altered form that causes a specific disease or pathology associated with the receptor. Both the GPCRs of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the receptor. These compounds can be further screened against a functional receptor to determine the effect of the compound on the receptor activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the receptor to a desired degree.

[0097] Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the receptor protein and a molecule that normally interacts with the receptor protein, e.g. a ligand or a component of the signal pathway that the receptor protein normally interacts (for example, a G-protein or other interactor involved in cAMP or phosphatidylinositol turnover and/or adenylate cyclase, or phospholipase C activation). Such assays typically include the steps of combining the receptor protein with a candidate compound under conditions that allow the receptor protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the receptor protein and the target, such as any of the associated effects of signal transduction such as G-protein phosphorylation, cAMP or phosphatidylinositol turnover, and adenylate cyclase or phospholipase C activation.

[0098] Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab′)₂, Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).

[0099] One candidate compound is a soluble fragment of the receptor that competes for ligand binding. Other candidate compounds include mutant receptors or appropriate fragments containing mutations that affect receptor function and thus compete for ligand. Accordingly, a fragment that competes for ligand, for example with a higher affinity, or a fragment that binds ligand but does not allow release, is encompassed by the invention.

[0100] The invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) receptor activity. The assays typically involve an assay of events in the signal transduction pathway that indicate receptor activity. Thus, a cellular process such as proliferation, the expression of genes that are up- or down-regulated in response to the receptor protein dependent signal cascade, can be assayed. In one embodiment, the regulatory region of such genes can be operably linked to a marker that is easily detectable, such as luciferase.

[0101] Any of the biological or biochemical functions mediated by the receptor can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly FIG. 2. Specifically, a biological function of a cell or tissues that expresses the receptor can be assayed. Experimental data as provided in FIG. 1 indicates that GPCR splice forms 1 and 2 of the present invention are expressed in head/neck tissue, infant brain, and stomach, as indicated by virtual northern blot analysis. Virtual northern blot analysis also indicates that splice form 2 is expressed in colon tumors and, additionally, PCR-based tissue screening panels indicate that splice form 2 is also expressed in leukocytes.

[0102] Binding and/or activating compounds can also be screened by using chimeric receptor proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions. For example, a G-protein-binding region can be used that interacts with a different G-protein then that which is recognized by the native receptor. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. Alternatively, the entire transmembrane portion or subregions (such as transmembrane segments or intracellular or extracellular loops) can be replaced with the entire transmembrane portion or subregions specific to a host cell that is different from the host cell from which the amino terminal extracellular domain and/or the G-protein-binding region are derived. This allows for assays to be performed in other than the specific host cell from which the receptor is derived. Alternatively, the amino terminal extracellular domain (and/or other ligand-binding regions) could be replaced by a domain (and/or other binding region) binding a different ligand, thus, providing an assay for test compounds that interact with the heterologous amino terminal extracellular domain (or region) but still cause signal transduction. Finally, activation can be detected by a reporter gene containing an easily detectable coding region operably linked to a transcriptional regulatory sequence that is part of the native signal transduction pathway.

[0103] The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the receptor. Thus, a compound is exposed to a receptor polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide (Hodgson, Bio/technology, Sep. 10, 1992, (9);973-80). Soluble receptor polypeptide is also added to the mixture. If the test compound interacts with the soluble receptor polypeptide, it decreases the amount of complex formed or activity from the receptor target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the receptor. Thus, the soluble polypeptide that competes with the target receptor region is designed to contain peptide sequences corresponding to the region of interest.

[0104] To perform cell free drug screening assays, it is sometimes desirable to immobilize either the receptor protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.

[0105] Techniques for immobilizing proteins on matrices can be used in the drug screening assays. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of receptor-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation. Preparations of a receptor-binding protein and a candidate compound are incubated in the receptor protein-presenting wells and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the receptor protein target molecule, or which are reactive with receptor protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

[0106] Agents that modulate one of the GPCRs of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.

[0107] Modulators of receptor protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the receptor pathway, by treating cells or tissues that express the GPCR. Experimental data as provided in FIG. 1 indicates that splice forms 1 and 2 are expressed in head/neck tissue, infant brain, and stomach. Splice form 2 is also expressed in colon tumors and leukocytes. These methods of treatment include the steps of administering a modulator of the GPCR's activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein.

[0108] In yet another aspect of the invention, the GPCR proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with the GPCR and are involved in GPCR activity. Such GPCR-binding proteins are also likely to be involved in the propagation of signals by the GPCR proteins or GPCR targets as, for example, downstream elements of a GPCR-mediated signaling pathway. Alternatively, such GPCR-binding proteins are likely to be GPCR inhibitors.

[0109] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a GPCR protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a GPCR-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional-regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the GPCR protein.

[0110] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a GPCR modulating agent, an antisense GPCR nucleic acid molecule, a GPCR-specific antibody, or a GPCR-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

[0111] The GPCR proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. Experimental data as provided in FIG. 1 indicates that splice forms 1 and 2 are expressed in head/neck tissue, infant brain, and stomach. Splice form 2 is also expressed in colon tumors and leukocytes. The method involves contacting a biological sample with a compound capable of interacting with the receptor protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

[0112] One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.

[0113] The peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs. Thus, the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered receptor activity in cell-based or cell-free assay, alteration in ligand or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

[0114] In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.

[0115] The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action. Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype. The discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the receptor protein in which one or more of the receptor functions in one population is different from those in another population. The peptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality. Thus, in a ligand-based treatment, polymorphism may give rise to amino terminal extracellular domains and/or other ligand-binding regions that are more or less active in ligand binding, and receptor activation. Accordingly, ligand dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism. As an alternative to genotyping, specific polymorphic peptides could be identified.

[0116] The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Experimental data as provided in FIG. 1 indicates that splice forms 1 and 2 are expressed in head/neck tissue, infant brain, and stomach. Splice form 2 is also expressed in colon tumors and leukocytes. Accordingly, methods for treatment include the use of the GPCR protein or fragments.

[0117] Antibodies

[0118] The invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof. As used herein, an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross-reactivity.

[0119] As used herein, an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge. The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0120] Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0121] In general, to generate antibodies, an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. The full-length protein, an antigenic peptide fragment or a fusion protein can be used. Particularly important fragments are those covering functional domains, such as the domains identified in FIG. 2, and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures.

[0122] Antibodies are preferably prepared from regions or discrete fragments of the GPCR proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or receptor/binding partner interaction. FIG. 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence fragments.

[0123] An antigenic fragment will typically comprise at least 8 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness (see FIG. 2).

[0124] Detection on an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

[0125] Antibody Uses

[0126] The antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Experimental data as provided in FIG. 1 indicates that GPCR splice forms 1 and 2 of the present invention are expressed in head/neck tissue, infant brain, and stomach, as indicated by virtual northern blot analysis. Virtual northern blot analysis also indicates that splice form 2 is expressed in colon tumors and, additionally, PCR-based tissue screening panels indicate that splice form 2 is also expressed in leukocytes. Further, such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover.

[0127] Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. Experimental data as provided in FIG. 1 indicates that splice forms 1 and 2 are expressed in head/neck tissue, infant brain, and stomach. Splice form 2 is also expressed in colon tumors and leukocytes. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein.

[0128] The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. Experimental data as provided in FIG. 1 indicates that splice forms 1 and 2 are expressed in head/neck tissue, infant brain, and stomach. Splice form 2 is also expressed in colon tumors and leukocytes. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.

[0129] Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.

[0130] The antibodies are also useful for tissue typing. Experimental data as provided in FIG. 1 indicates that splice forms 1 and 2 are expressed in head/neck tissue, infant brain, and stomach. Splice form 2 is also expressed in colon tumors and leukocytes. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.

[0131] The antibodies are also useful for inhibiting protein function, for example, blocking the binding of the GPCR peptide to a binding partner such as a ligand. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See FIG. 2 for structural information relating to the proteins of the present invention.

[0132] The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nucleic acid arrays and similar methods have been developed for antibody arrays.

[0133] Nucleic Acid Molecules

[0134] The present invention further provides isolated nucleic acid molecules that encode a GPCR peptide or protein of the present invention (cDNA, transcript and genomic sequence). Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the GPCR peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof.

[0135] As used herein, an “isolated” nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.

[0136] Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.

[0137] For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include, such molecules produced synthetically.

[0138] Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NOS:1 and 4, transcript/cDNA sequence, and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NOS:2 and 5. A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.

[0139] The present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NOS:1 and 4, transcript/cDNA sequence, and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.

[0140] The present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NOS:1 and 4, transcript/cDNA sequence, and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the proteins provided in FIG. 2, SEQ ID NOS:2 and 5. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences. Such a nucleic acid molecule can have a few additional nucleotides or can comprises several hundred or more additional nucleotides. A brief description of how various types of these nucleic acid molecules can be readily made/isolated is provided below.

[0141] In FIGS. 1 and 3, both coding and non-coding sequences are provided. Because of the source of the present invention, human genomic sequences (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5′ and 3′ non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in FIGS. 1 and 3 or can readily be identified using computational tools known in the art. As discussed below, some of the non-coding regions, particularly gene regulatory elements such as promoters, are useful for a variety of purposes, e.g. control of heterologous gene expression, target for identifying gene activity modulating compounds, and are particularly claimed as fragments of the genomic sequence provided herein.

[0142] The isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.

[0143] As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the GPCR peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5′ and 3′ sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.

[0144] Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).

[0145] The invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the GPCR proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.

[0146] The present invention further provides non-coding fragments of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents. A promoter can readily be identified as being 5′ to the ATG start site in the genomic sequence provided in FIG. 3.

[0147] A fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene.

[0148] A probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.

[0149] Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence shown in the Figure sheets or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene. The gene encoding the novel GPCR proteins of the present invention is located on a genome component that has been mapped to human chromosome 1 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

[0150]FIG. 3 provides information on SNPs that have been found in the gene encoding the GPCR proteins of the present invention. SNPs were identified at 107 different nucleotide positions. Some of these SNPs that are located outside the ORF and in introns may affect gene expression.

[0151] As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45 C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65 C. Examples of moderate to low stringency hybridization conditions are well known in the art.

[0152] Nucleic Acid Molecule Uses

[0153] The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in FIG. 2 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in FIG. 2. As illustrated in FIG. 3, SNPs were identified at 107 different nucleotide positions.

[0154] The probe can correspond to any sequence along the entire length of the nucleic acid molecules provided in the Figures. Accordingly, it could be derived from 5′ noncoding regions, the coding region, and 3′ noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention.

[0155] The nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.

[0156] The nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product. For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.

[0157] The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.

[0158] The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods. The gene encoding the novel GPCR proteins of the present invention is located on a genome component that has been mapped to human chromosome 1 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

[0159] The nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.

[0160] The nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.

[0161] The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.

[0162] The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.

[0163] The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.

[0164] The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Experimental data as provided in FIG. 1 indicates that GPCR splice forms 1 and 2 of the present invention are expressed in head/neck tissue, infant brain,, and stomach, as indicated by virtual northern blot analysis. Virtual northern blot analysis also indicates that splice form 2 is expressed in colon tumors and, additionally, PCR-based tissue screening panels indicate that splice form 2 is also expressed in leukocytes. Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in GPCR protein expression relative to normal results.

[0165] In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA includes Southern hybridizations and in situ hybridization.

[0166] Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a GPCR protein, such as by measuring a level of a receptor-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a receptor gene has been mutated. Experimental data as provided in FIG. 1 indicates that GPCR splice forms 1 and 2 of the present invention are expressed in head/neck tissue, infant brain, and stomach, as indicated by virtual northern blot analysis. Virtual northern blot analysis also indicates that splice form 2 is expressed in colon tumors and, additionally, PCR-based tissue screening panels indicate that splice form 2 is also expressed in leukocytes.

[0167] Nucleic acid expression assays are useful for drug screening to identify compounds that modulate GPCR nucleic acid expression.

[0168] The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the GPCR gene, particularly biological and pathological processes that are mediated by the GPCR in cells and tissues that express it. Experimental data as provided in FIG. 1 indicates that splice forms 1 and 2 are expressed in head/neck tissue, infant brain, and stomach. Splice form 2 is also expressed in colon tumors and leukocytes. The method typically includes assaying the ability of the compound to modulate the expression of the GPCR nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired GPCR nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the GPCR nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.

[0169] The assay for GPCR nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the GPCR protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.

[0170] Thus, modulators of GPCR gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of GPCR mRNA in the presence of the candidate compound is compared to the level of expression of GPCR mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.

[0171] The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate GPCR nucleic acid expression, particularly to modulate activities within a cell or tissue that expresses the proteins. Experimental data as provided in FIG. 1 indicates that GPCR splice forms 1 and 2 of the present invention are expressed in head/neck tissue, infant brain, and stomach, as indicated by virtual northern blot analysis. Virtual northern blot analysis also indicates that splice form 2 is expressed in colon tumors and, additionally, PCR-based tissue screening panels indicate that splice form 2 is also expressed in leukocytes. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

[0172] Alternatively, a modulator for GPCR nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the GPCR nucleic acid expression in the cells and tissues that express the protein. Experimental data as provided in FIG. 1 indicates that splice forms 1 and 2 are expressed in head/neck tissue, infant brain, and stomach. Splice form 2 is also expressed in colon tumors and leukocytes.

[0173] The nucleic acid molecules are also usefull for monitoring the effectiveness of modulating compounds on the expression or activity of the GPCR gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.

[0174] The nucleic acid molecules are also useful in diagnostic assays for qualitative changes in GPCR nucleic acid, and particularly in qualitative changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in GPCR genes and gene expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally-occurring genetic mutations in the GPCR gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the GPCR gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a GPCR protein.

[0175] Individuals carrying mutations in the GPCR gene can be detected at the nucleic acid level by a variety of techniques. FIG. 3 provides information on SNPs that have been found in the gene encoding the GPCR proteins of the present invention. SNPs were identified at 107 different nucleotide positions. Some of these SNPs that are located outside the ORF and in introns may affect gene expression. The gene encoding the novel GPCR proteins of the present invention is located on a genome component that has been mapped to human chromosome 1 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.

[0176] Alternatively, mutations in a GPCR gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.

[0177] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature.

[0178] Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S1 protection or the chemical cleavage method. Furthermore, sequence differences between a mutant GPCR gene and a wild-type gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).

[0179] Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et al., Nature 313:495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.

[0180] The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the GPCR gene in an individual in order to select an appropriate compound or dosage regimen for treatment. As illustrated in FIG. 3, SNPs were identified at 107 different nucleotide positions.

[0181] Thus nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.

[0182] The nucleic acid molecules are thus useful as antisense constructs to control GPCR gene expression in cells, tissues, and organisms. A DNA antisense nucleic acid molecule is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of GPCR protein. An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into GPCR protein.

[0183] Alternatively, a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of GPCR nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired GPCR nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the GPCR protein, such as ligand binding.

[0184] The nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in GPCR gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired GPCR protein to treat the individual.

[0185] The invention also encompasses kits for detecting the presence of a GPCR nucleic acid in a biological sample. Experimental data as provided in FIG. 1 indicates that GPCR splice forms 1 and 2 of the present invention are expressed in head/neck tissue, infant brain, and stomach, as indicated by virtual northern blot analysis. Virtual northern blot analysis also indicates that splice form 2 is expressed in colon tumors and, additionally, PCR-based tissue screening panels indicate that splice form 2 is also expressed in leukocytes. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting GPCR nucleic acid in a biological sample; means for determining the amount of GPCR nucleic acid in the sample; and means for comparing the amount of GPCR nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect GPCR protein mRNA or DNA.

[0186] Nucleic Acid Arrays

[0187] The present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1, 3, and 4).

[0188] As used herein “Arrays” or “Microarrays” refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT application W095/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et. al., U.S. Pat. No. 5,807,522.

[0189] The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5′, or 3′, sequence, sequential oligonucleotides which cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.

[0190] In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5′ or at the 3′ end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The “pairs” will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.

[0191] In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a “gridded” array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.

[0192] In order to conduct sample analysis using a microarray or detection kit, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit. The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.

[0193] Using such arrays, the present invention provides methods to identify the expression of the GPCR proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the GPCR gene of the present invention. FIG. 3 provides information on SNPs that have been found in the gene encoding the GPCR proteins of the present invention. SNPs were identified at 107 different nucleotide positions. Some of these SNPs that are located outside the ORF and in introns may affect gene expression.

[0194] Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0195] The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.

[0196] In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.

[0197] Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.

[0198] In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified GPCR genes of the present invention can be routinely identified using the sequence information disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.

[0199] Vectors/Host Cells

[0200] The invention also provides vectors containing the nucleic acid molecules described herein. The term “vector” refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0201] A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.

[0202] The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules. The vectors can function in procaryotic or eukaryotic cells or in both (shuttle vectors).

[0203] Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.

[0204] The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.

[0205] In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0206] In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0207] A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, eg. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0208] The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.

[0209] The nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.

[0210] The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

[0211] As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).

[0212] Recombinant protein expression can be maximized in a host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli. (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0213] The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

[0214] The nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

[0215] In certain embodiments of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)).

[0216] The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0217] The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).

[0218] The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.

[0219] The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0220] Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the nucleic acid molecule vector.

[0221] In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.

[0222] Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.

[0223] While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.

[0224] Where secretion of the peptide is desired, which is difficult to achieve with multi-transmembrane domain containing proteins such as GPCRs, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides.

[0225] Where the peptide is not secreted into the medium, which is typically the case with GPCRs, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.

[0226] It is also understood that depending upon the host cell in recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.

[0227] Uses of Vectors and Host Cells

[0228] The recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing a GPCR protein or peptide that can be further purified to produce desired amounts of GPCR protein or fragments. Thus, host cells containing expression vectors are useful for peptide production.

[0229] Host cells are also useful for conducting cell-based assays involving the GPCR protein or GPCR protein fragments, such as those described above as well as other formats known in the art. Thus, a recombinant host cell expressing a native GPCR protein is useful for assaying compounds that stimulate or inhibit GPCR protein function.

[0230] Host cells are also useful for identifying GPCR protein mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant GPCR protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native GPCR protein.

[0231] Genetically engineered host cells can be further used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a GPCR protein and identifying and evaluating modulators of GPCR protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.

[0232] A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the GPCR protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.

[0233] Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the GPCR protein to particular cells.

[0234] Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.

[0235] In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992). Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

[0236] Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. Nature 385:810-813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G_(o) phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

[0237] Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect ligand binding, GPCR protein activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo GPCR protein function, including ligand interaction, the effect of specific mutant GPCR proteins on GPCR protein function and ligand interaction, and the effect of chimeric GPCR proteins. It is also possible to assess the effect of null mutations, that is mutations that substantially or completely eliminate one or more GPCR protein functions.

[0238] All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

1 7 1 2487 DNA Human 1 atgcgcttgg agggagaggg ccgctcagcg agggcgggac agaacctctc ccgggctggg 60 agtgcacggc gcggtgcgcc cagggacctc agcatgaaca acctcacaga gcttcagcct 120 ggcctcttcc accacctgcg cttcttggag gagctgcgtc tctctgggaa ccatctctca 180 cacatcccag gacaagcatt ctctggtctc tacagcctga aaatcctgat gctgcagaac 240 aatcagctgg gaggaatccc cgcagaggcg ctgtgggagc tgccgagcct gcagtcgcta 300 gacctgaatt ataacaagct gcaggagttc cctgtggcca tccggaccct gggcagactg 360 caggaactgg ggttccataa caacaacatc aaggccatcc cagaaaaggc cttcatgggg 420 aaccctctgc tacagacgat acacttttat gataacccaa tccagtttgt gggaagatcg 480 gcattccagt acctgcctaa actccacaca ctatctctga atggtgccat ggacatccag 540 gagtttccag atctcaaagg caccaccagc ctggagatcc tgaccctgac ccgcgcaggc 600 atccggctgc tcccatcggg gatgtgccaa cagctgccca ggctccgagt cctggaactg 660 tctcacaatc aaattgagga gctgcccagc ctgcacaggt gtcagaaatt ggaggaaatc 720 ggcctccaac acaaccgcat ctgggaaatt ggagctgaca ccttcagcca gctgagctcc 780 ctgcaagccc tggatcttag ctggaacgcc atccggtcca tccaccccga ggccttctcc 840 accctgcact ccctggtcaa gctggacctg acagacaacc agctgaccac actgcccctg 900 gctggacttg ggggcttgat gcatctgaag ctcaaaggga accttgctct ctcccaggcc 960 ttctccaagg acagtttccc aaaactgagg atcctggagg tgccttatgc ctaccagtgc 1020 tgtccctatg ggatgtgtgc cagcttcttc aaggcctctg ggcagtggga ggctgaagac 1080 cttcaccttg atgatgagga gtcttcaaaa aggcccctgg gcctccttgc cagacaagca 1140 gagaaccact atgaccagga cctggatgag ctccagctgg agatggagga ctcaaagcca 1200 caccccagtg tccagtgtag ccctactcca ggccccttca agccctgtga gtacctcttt 1260 gaaagctggg gcatccgcct ggccgtgtgg gccatcgtgt tgctctccgt gctctgcaat 1320 ggactggtgc tgctgaccgt gttcgctggc gggcctgccc ccctgccccc ggtcaagttt 1380 gtggtaggtg cgattgcagg cgccaacacc ttgactggca tttcctgtgg ccttctagcc 1440 tcagtcaatg ccctgacctt tggtcagttc tctgagtacg gagcccgctg ggagacgggg 1500 ctaggctgcc gggccactgg cttcctggca gtacttgggt cggaggcatc ggtgctgctg 1560 ctcactctgg ccgcagtgca gtgcagcgtc tccgtctcct gtgtccgggc ctatgggaag 1620 tccccctccc tgggcagcgt tcgagcaggg gtcctaggct gcctggcact ggcagggctg 1680 gccgccgcac tgcccctggc ctcagtggga gaatacgggg cctccccact ctgcctgccc 1740 tacgcgccac ctgagggtca gccagcagcc ctgggcttca ccgtggccct ggtgatgatg 1800 aactccttct gtttcctggt cgtggccggt gcctacatca aactgtactg tgacctgccg 1860 cggggcgact ttgaggccgt gtgggactgc gccatggtga ggcacgtggc ctggctcatc 1920 ttcgcagacg ggctcctcta ctgtcccgtg gccttcctca gctttgcctc catgctgggc 1980 ctcttccctg tcacgcccga ggccgtcaag tctgtcctgc tggtggtgct gcccctgcct 2040 gcctgcctca acccactgct gtacctgctc ttcaaccccc acttccggga tgaccttcgg 2100 cggcttcggc cccgcgcagg ggactcaggg cccctagcct atgctgcggc cggggagctg 2160 gagaagagct cctgtgattc tacccaggcc ctggtagcct tctctgatgt ggatctcatt 2220 ctggaagctt ctgaagctgg gcggccccct gggctggaga cctatggctt cccctcagtg 2280 accctcatct cctgtcagca gccaggggcc cccaggctgg agggcagcca ttgtgtagag 2340 ccagagggga accactttgg gaacccccaa ccctccatgg atggagaact gctgctgagg 2400 gcagagggat ctacgccagc aggtggaggc ttgtcagggg gtggcggctt tcagccctct 2460 ggcttggcct ttgcttcaca cgtgtaa 2487 2 828 PRT Human 2 Met Arg Leu Glu Gly Glu Gly Arg Ser Ala Arg Ala Gly Gln Asn Leu 1 5 10 15 Ser Arg Ala Gly Ser Ala Arg Arg Gly Ala Pro Arg Asp Leu Ser Met 20 25 30 Asn Asn Leu Thr Glu Leu Gln Pro Gly Leu Phe His His Leu Arg Phe 35 40 45 Leu Glu Glu Leu Arg Leu Ser Gly Asn His Leu Ser His Ile Pro Gly 50 55 60 Gln Ala Phe Ser Gly Leu Tyr Ser Leu Lys Ile Leu Met Leu Gln Asn 65 70 75 80 Asn Gln Leu Gly Gly Ile Pro Ala Glu Ala Leu Trp Glu Leu Pro Ser 85 90 95 Leu Gln Ser Leu Asp Leu Asn Tyr Asn Lys Leu Gln Glu Phe Pro Val 100 105 110 Ala Ile Arg Thr Leu Gly Arg Leu Gln Glu Leu Gly Phe His Asn Asn 115 120 125 Asn Ile Lys Ala Ile Pro Glu Lys Ala Phe Met Gly Asn Pro Leu Leu 130 135 140 Gln Thr Ile His Phe Tyr Asp Asn Pro Ile Gln Phe Val Gly Arg Ser 145 150 155 160 Ala Phe Gln Tyr Leu Pro Lys Leu His Thr Leu Ser Leu Asn Gly Ala 165 170 175 Met Asp Ile Gln Glu Phe Pro Asp Leu Lys Gly Thr Thr Ser Leu Glu 180 185 190 Ile Leu Thr Leu Thr Arg Ala Gly Ile Arg Leu Leu Pro Ser Gly Met 195 200 205 Cys Gln Gln Leu Pro Arg Leu Arg Val Leu Glu Leu Ser His Asn Gln 210 215 220 Ile Glu Glu Leu Pro Ser Leu His Arg Cys Gln Lys Leu Glu Glu Ile 225 230 235 240 Gly Leu Gln His Asn Arg Ile Trp Glu Ile Gly Ala Asp Thr Phe Ser 245 250 255 Gln Leu Ser Ser Leu Gln Ala Leu Asp Leu Ser Trp Asn Ala Ile Arg 260 265 270 Ser Ile His Pro Glu Ala Phe Ser Thr Leu His Ser Leu Val Lys Leu 275 280 285 Asp Leu Thr Asp Asn Gln Leu Thr Thr Leu Pro Leu Ala Gly Leu Gly 290 295 300 Gly Leu Met His Leu Lys Leu Lys Gly Asn Leu Ala Leu Ser Gln Ala 305 310 315 320 Phe Ser Lys Asp Ser Phe Pro Lys Leu Arg Ile Leu Glu Val Pro Tyr 325 330 335 Ala Tyr Gln Cys Cys Pro Tyr Gly Met Cys Ala Ser Phe Phe Lys Ala 340 345 350 Ser Gly Gln Trp Glu Ala Glu Asp Leu His Leu Asp Asp Glu Glu Ser 355 360 365 Ser Lys Arg Pro Leu Gly Leu Leu Ala Arg Gln Ala Glu Asn His Tyr 370 375 380 Asp Gln Asp Leu Asp Glu Leu Gln Leu Glu Met Glu Asp Ser Lys Pro 385 390 395 400 His Pro Ser Val Gln Cys Ser Pro Thr Pro Gly Pro Phe Lys Pro Cys 405 410 415 Glu Tyr Leu Phe Glu Ser Trp Gly Ile Arg Leu Ala Val Trp Ala Ile 420 425 430 Val Leu Leu Ser Val Leu Cys Asn Gly Leu Val Leu Leu Thr Val Phe 435 440 445 Ala Gly Gly Pro Ala Pro Leu Pro Pro Val Lys Phe Val Val Gly Ala 450 455 460 Ile Ala Gly Ala Asn Thr Leu Thr Gly Ile Ser Cys Gly Leu Leu Ala 465 470 475 480 Ser Val Asn Ala Leu Thr Phe Gly Gln Phe Ser Glu Tyr Gly Ala Arg 485 490 495 Trp Glu Thr Gly Leu Gly Cys Arg Ala Thr Gly Phe Leu Ala Val Leu 500 505 510 Gly Ser Glu Ala Ser Val Leu Leu Leu Thr Leu Ala Ala Val Gln Cys 515 520 525 Ser Val Ser Val Ser Cys Val Arg Ala Tyr Gly Lys Ser Pro Ser Leu 530 535 540 Gly Ser Val Arg Ala Gly Val Leu Gly Cys Leu Ala Leu Ala Gly Leu 545 550 555 560 Ala Ala Ala Leu Pro Leu Ala Ser Val Gly Glu Tyr Gly Ala Ser Pro 565 570 575 Leu Cys Leu Pro Tyr Ala Pro Pro Glu Gly Gln Pro Ala Ala Leu Gly 580 585 590 Phe Thr Val Ala Leu Val Met Met Asn Ser Phe Cys Phe Leu Val Val 595 600 605 Ala Gly Ala Tyr Ile Lys Leu Tyr Cys Asp Leu Pro Arg Gly Asp Phe 610 615 620 Glu Ala Val Trp Asp Cys Ala Met Val Arg His Val Ala Trp Leu Ile 625 630 635 640 Phe Ala Asp Gly Leu Leu Tyr Cys Pro Val Ala Phe Leu Ser Phe Ala 645 650 655 Ser Met Leu Gly Leu Phe Pro Val Thr Pro Glu Ala Val Lys Ser Val 660 665 670 Leu Leu Val Val Leu Pro Leu Pro Ala Cys Leu Asn Pro Leu Leu Tyr 675 680 685 Leu Leu Phe Asn Pro His Phe Arg Asp Asp Leu Arg Arg Leu Arg Pro 690 695 700 Arg Ala Gly Asp Ser Gly Pro Leu Ala Tyr Ala Ala Ala Gly Glu Leu 705 710 715 720 Glu Lys Ser Ser Cys Asp Ser Thr Gln Ala Leu Val Ala Phe Ser Asp 725 730 735 Val Asp Leu Ile Leu Glu Ala Ser Glu Ala Gly Arg Pro Pro Gly Leu 740 745 750 Glu Thr Tyr Gly Phe Pro Ser Val Thr Leu Ile Ser Cys Gln Gln Pro 755 760 765 Gly Ala Pro Arg Leu Glu Gly Ser His Cys Val Glu Pro Glu Gly Asn 770 775 780 His Phe Gly Asn Pro Gln Pro Ser Met Asp Gly Glu Leu Leu Leu Arg 785 790 795 800 Ala Glu Gly Ser Thr Pro Ala Gly Gly Gly Leu Ser Gly Gly Gly Gly 805 810 815 Phe Gln Pro Ser Gly Leu Ala Phe Ala Ser His Val 820 825 3 119596 DNA Human misc_feature (1)...(119596) n = A,T,C or G 3 ccccatttcc ctcccatcca ccagcagtgg tttctgggag cccatctatg ggggctactt 60 ggaggggaag gtattggggc tcagcacaga aattgcggac tctggggagg gtaagacgga 120 atccttgagt gtggagatta tcatcagcgt tgatgttgag gacctactgt ctgctgggtc 180 ctctgggagg cagtcagggt gatgactgtc agtgagagac cagccaggtg cacgtgagaa 240 gtgaactcac tgtatcagat aatcctggga gcatccaggg aggaccagag gtgaggtcca 300 caggcattca agggagaaag aggaccgagg tggtcaggga aggcccaggg gagagagaag 360 aggcttgagt cctgaagacc agaaggactt aactcagggc caggagaagg ggaagccatg 420 agcccaggtt agaatgagga accacattgg ggcttttgta cttcatctcc acgacctgtt 480 ctgtcccact ccctggggca gcatgttcta gagcagggtt ttctgccctt tttccctcgg 540 ggcacacatt ttaaaaacac agacacacac atacacaaac acacaaacgc aatccaaggt 600 attcagtgtc ctgggttaaa ccaacaaggc ctctcaggcg aaaggcaccc agtcaaccct 660 gggcagcatc cagccatccc aagggacagg agaccccatc tgggcacaca cctggaagcg 720 ctgctataga agaaagagtg catgtttgca gccaagcaga catgggttcg aggctaagtt 780 ccagctctaa ccagctatgg ctatatgttt cttcacctct ctcagcttct gttttcttgc 840 tttcaaaatg ggcatacgga aagatgttta taattagaca cagtgcttgg cacatagcaa 900 atgcccaatc tatgggagct atggttatta tctatctagt ctccccttga gaggaggcac 960 ttgggaggag cggtggaggc aggcctgcag tggcccttca tttcacctat ttgactctgc 1020 ctgcctagag cttgccgaga atgagaccca gagggtcggt cctaggacac aattcctctc 1080 accctctgct tctgccacca tcctttcccc tttgcccttg gaggtgctgg aggaggtggg 1140 agtgtccccg agagtgtcca gactgcataa ggtggcagaa aggtgctgag tgtcttccat 1200 tgcaccagtg tcagggactt ggatgtggtc cccaggccca aagccaaggc agatggactc 1260 atggaggggg aggagcagag gagcagtgcg gagtcactct ctccatgtag cacccccagc 1320 tcagtgcatt gctggggttc ttgcagggca gatgaagtgc ttctttgtat aggggaatat 1380 gtgggtgaac caggaaaatg ggagcctagt acatggaggt ttattccaac tccctgtgtg 1440 attggcaagt tagaattgcc cccctacctg agttattatt ggctcctgag aaacaggcca 1500 aaggcagccc ccactaagaa gcttttagca cctcctatag gaaggaagcc ttcccagtga 1560 atccagtgtg aggattcttt tctccaggtc tctggacctc tcccaacatg ctccatgttc 1620 ctttgcacga agaagaacag cgcagttcgg tagagtgtat gtaagccccg gttgccatca 1680 gcacactgtg tggattctgg caatacctgt catccccact ggtgctgcgg tgcccctcat 1740 gccccttgtt aataaccaat gatagtgcca cttgaactcc caggctgttg tgaggagcaa 1800 gcattcattc attcattcat tcaatcactc atttgttcat gtgacaaatg tggattgaat 1860 gcttgccctc tttcctctcc cctggagcct tggagagaga gataagacaa gcgcaaataa 1920 ctgtgacctg ttgtaggaag tgagaaatac acccaggcag ggccagatac tgcgtagcag 1980 gcggggcaca gaggagggaa cgcttgtgtc taactgatgc tcctaatgcg gaagcccctg 2040 aaaggcggtt gtggtgcaaa ggaaaaccca caggccaagg aatgggaaga ccaaggttga 2100 cacttgtttg tcaagtgtca ataatcatct ctgcccggtg agttgttgca tgaagcaaga 2160 tcctccttgg ggggtgttta gcacagaggg ggtgcgattt tatttcctgt gaaaatacca 2220 ggtagtatta tgttcactat gtctttgtag ggtgtgtgtg tccccatctg tctgtatgtt 2280 tgtctgattg tgatgctcta cttcatcaga ttcagggttc cttgtggaca ggagctgtgt 2340 ttctttgcat caggagaggg gaactctttt agggcagggg ccatgcctcc tccttcagtc 2400 tgggagcttc ctgaaggcag gatctgtgct cgtgggtcct tttcttggga tcacatcaca 2460 gacacctcct ttcagcacac acacacacct ccacacacac acacacctcc acacacaagc 2520 tctaaacaca cacacgcctc caaacacaca cacgtgcaaa cacacacaca cccgacacac 2580 gcctcctcct aacacacaca cctccacaca cacacacctc cacacacaca cacctccaca 2640 cacacacctc cacacacaca cctccaaaca cacaccacac acacacctcc aaacacacac 2700 acacacctcc acacacacac acctccacac acacacctcc aaacacacct ccacacacac 2760 acctccaaac acacacacac ctccaaacac acacacctcc ttcaaacaca cacacacctc 2820 caaacacaaa cacacctaac acacacctcc ttcaaacaca cacacctcca aacacacacc 2880 tccttcaaac acacacacct ccaaacacac acacacctcc aaacacacac acgcctccaa 2940 acacacacac acctctccaa aaacacacac acacctaaca cacacctcct tcaaacacac 3000 acacacctcc aaacacaaac acacctaaca cacccctcct tcaaacacac acacctcaaa 3060 acacacacac ctcaaaacac acacacctcc aaaaacacac acatacctaa cacacacctc 3120 cttcaaacac acacacctcc aaacacacac acacctaaca cacacctgtt tgaaacacac 3180 acacacctgc acacacacct aacacacact tccttcaaac acacaccctc gaaacacatg 3240 tccttcaaac acacacacac cctccaaaca cacacacctc cttcaaacac acacacacac 3300 ctccaaacac acacacacac ctaacacaca cctccttcaa gcacacacac gcacctccaa 3360 acacacacac acctaacaca cacctcctcc aaacacacac acacctccaa acacacacac 3420 ctcacacacc tccttcaaac acacacacct ccaaacacac acacacctaa cacacacctc 3480 ctccacacac acacacgtac ctccaaacaa acacacacct aacacacctc cttcaaacac 3540 acacacacct ccacacacac ctaacacaca cttccttcaa acacacaccc tcgaaacaca 3600 tgtccttcaa acacacacac accctccaaa cacacacacc tccttcaaac acacacacac 3660 acctccaaac acacacacac gcctaacaca cacctccttc aagcacacac acgcacctcc 3720 aaacacacac acacctaaca cgcctccttc aaacacacac acacctccaa acacacacac 3780 ctcacacacc tccttcaaac acacacacct ccaaacacac acacacctaa cacacacctc 3840 cttcaaacac acacacacct ccaaacacac acacacctcc ttcaaacaca cacacaccct 3900 gcttcaaaca cacacaccct ccttcaagca cacacacaca cctccaaaca catatgcaca 3960 cctccttcaa acatacacat acacaccctc cttcaaacat acacacacac ctccttcaca 4020 catacacata cacctccttc aaacacacac acacacgcac acctccctca aacatacaca 4080 cacacctcct tcaaacatac acacacaccc ctaacacaca caccttcttc aaacacacac 4140 acacacctcc ttcaaataca cacacacacc tccaaacaca tacgcacacc tccttcaaac 4200 atacacacct ccttcataca cacacacccc aacacacaca cctccttcaa acacacacac 4260 ctccttcaaa cacacacccg tccttcaaac acacacacac ctaacacaca cacacacctc 4320 cttcaaacat acacacacat ccttcaagaa cacacacaca cacagacaca caacacacac 4380 acacacacac acacacccct agtatgggtc tccgcctgca gggggcgctc aggacaggac 4440 tgtaaacagt tccagtgtat gcatttcatt ttttctccct tcctctcagc tccagatctg 4500 gggcaaccca gggttgggag tgtttgagga tggaggacac tggatcaagt gtccaggaat 4560 tgtgagagtt gaggcagtta ggggctgtaa actaatccgc tgcctttgtg gtggacagag 4620 cagattagga gactgggagg agacgagaaa ggaagggaac cattgtctag gacaaaagca 4680 agagagaatg agcattgcga caggacctgg gctcccaccc tcacttacct ggggccgatg 4740 agagctcagg acccaaagga gtcagagccg ggaaccagat cacgtctcag gccaccatgg 4800 acccggcccc ccagcgcagt tacctgcttc agcatgtgtg actgggtggc caggatccct 4860 tcacagacat gccacccaca cagtcacaca gggccctgtg cttgggccat tgctctgctg 4920 ttgccatgtt atttttttcg gttgtgtgtg tgtgttattt tattttattt tttcgagaca 4980 ggatcttgca ctgtcacccc ggctggagtg caatggtacg atctccgctc actgcagcct 5040 caactccctg ggctcaagtg atcctcccac ttcagcctcc tgagtagccg agactacagg 5100 tgcacaccac cacaaccagc taatttttaa gtttttttaa aattaaaaaa cttaaaactt 5160 atagtgggat ctcactatat tgcccaggct ggtcttgaac ttgctgggat tacaggcgag 5220 agagctgtgt aaatttttaa taattttatt acaaggggcc ccacattttt attttgcact 5280 gggtgctgca aattatttta tttatttatt tatttattta tttatttatt tatttattga 5340 gacagagtct cgctctgtca cccaggctgg agtgcaatgg cgctatcttg gctcactgca 5400 acctccgcct ctcgggttca agcgattctt ctgcctcagc ctctcaagta gctgggatta 5460 taggctcctg ccaccaagcc cagctaattt ttgtattttt agtagagacg ggttttcgcc 5520 atgtggccag gctggtctcg aactcctgac ctcaggtgat ccacccacct tgacctccca 5580 aagtgctggg attacaggtg tgagccactg tgcccggccg gtcctgcaaa ttatggagcc 5640 ctttctcccc ttaacctagg ggctagggca ggaggggatg agaggaaggg gtctccctct 5700 cagggatgcc acaggggaga tgaggcagcc tttgtccaga tgggcaggag gtggccccag 5760 gactagaggg gagactgagc aaccttgggt tggaggtgct tcttggcctg agcgtggcat 5820 ctgcaggggc cagggactat ggggtgcatt gtgctagagt gggcacaggg ggcacaccag 5880 agtctcagtg tttctgggaa tgaggcagtt gtcaggctct ggcaggcagg gattctgctt 5940 gaaagggagg gcagagggcc catcccacag atgtcagatc ctgtgtgtgt tggcctcctt 6000 cccagtttct tataaatcta gcagagtggg taagtgcaca aaccccagag ccactccacc 6060 taagttcaga ttctggctct gccttttacc aatccttggg gaaagattta ccctctttgt 6120 gctttagttt cctcacctgt aatagaggga tgacaacgat gcccttgtta aatgagttaa 6180 tatatgcaaa gtgcctagaa cggcgtctgg cacacagtaa ccattcaatg gatgttagct 6240 attatttttc atttattcaa cctgtgaata tgtattaagt atacacttag tagtaggcac 6300 agctgtgcta ggtgctgaca ctaactttca ctgcaaaaaa cagagtccct ggtccagctg 6360 tggagataaa ttcttagtaa cgccccaagt gagcgcctgg acttgggaaa tcctaccact 6420 gacctgcaga gccctgctgg ctcagcctct ccctccaatc tcccaccctc ctgctgcagt 6480 ttttctctgt cctgtggagt gaggagttgc atgagtcctc tggaggaagg aggtgcttcc 6540 aaagtttccc tcctccaatt tcgtcgcaac cctcctgact ccagcaactg gaactttagg 6600 tttgttctct ggtggaagtg ggatcaggag accctgggct aggacgctgt ctttgccgcc 6660 aacatgctga gtgcctttgg acaagtcctc tgatgccttt gggtctgttt cctgtggggt 6720 gagagttcct gaccccagga tattacagag acaaatgatg gtgtataaag aaagatacaa 6780 agaggggaga gagagaggag aaggaggggg gagagagaga gagaaggggc atgagtgagc 6840 caatgaagag gagactgtga tgtgagggag gtgccagggt cccctgagat ttgcagtgtt 6900 cactccgtga gacttgagat cccaggggcc gggataggac atggccatca tcagcacctc 6960 gagggctgtg tgcctaccca gaacaaggcc ccccattaac tatgtgcagc cagtggaaag 7020 aggaatgcct acccccccgc cagccgacgc cacgccagcc ctcgcctcgt tgcccactcc 7080 cctactctga atgcagaagc ccagggtgcc acctccccct ctgcctgccc tgcctccacc 7140 tgccagaaag cattagcttc actgtctaga ttggcatggg ggcgcaaagg caccatgttg 7200 cggggcccag ggcacctgct gggggtgggg cttggggtca ggaaatacct gggctaaccc 7260 ctggaaccct agcatgccca gtgccaatga gcaaatggcc ctagagccat aggggtaaag 7320 agtgtagtaa gaggggaagg cttggaagct cagaaacttg gcctggggag cccttctgac 7380 accttactcc agccccttac ccctaacctc ggctgggacc tccagatggg cagatcaagt 7440 cattacaccc caccatttgc ttagtgtttg accccctggg aatgtgatca ctgggaagtt 7500 cttcctagag tccctcctcc ttcctatcat agtgtcccca agtttccctc ctattttggc 7560 cagaggggag agagcccaaa gctaacccct tctttgttag ccccataggc aatggtgggg 7620 tgacctgggt ttcatttcac tttctccatg aggaagggat cctggcctat aaaggcaacc 7680 ttggagacgc cacacctgcc atccccaggg atcctggagg ggctgcctgc tgctgaggag 7740 gtcagaggcc tctccctgct cctcctcaga aatccacaca tccactcagc ctgcccgccc 7800 cacagtgttc tgggctgcag ctagtccagg caacatcttt gtgagaaata gaaaagggtg 7860 ccccttgccc cagtggccct accacgtggg gagcgggcag agttgatgag tggggcagga 7920 gcagattttg tcctctggtt ggccccttgg ccagacacct ttgtctagac caggccacag 7980 tggcatattt ctatgagtca gcctggtcaa tacttagtta aaaaaagtga tttaaaacat 8040 atttataaaa gattgcttaa aggaggttta ttctatggat tcccttggat atcctaaagc 8100 ctttcactcg aagtatggtt cacagcccag cagcatagca tcggcatcat ctgtgagtgt 8160 gtgaggaatg cagaagctca ggcctcaacc ctgacctact gagccagaat cttcatttta 8220 gggtttgcca agcactgcct caagcttatt catggaataa gtcagatcat cagtggagct 8280 gctggacagc cactctctac tccatctcct acagaaatgc ttttacctag tttgtactta 8340 ggtgatgcac cattggttta tatccaggtc tataagaaac agccctatgg acatcacggt 8400 tgatttactg caaggatgtg gtgaagaaat gggttcagac cagaagcctg ctgctctggt 8460 ggggtatttg gaagaggcaa ggaagcatca gcttcattta ggatgtaagg ggctgactga 8520 gatgtctctt cctctgggaa accttcccta atccctcaac tctgggggag gcctcccctc 8580 tgtgtgcctg ccctgccgca aggctcattc ccttgggttc agattcctgg ttacttgtct 8640 gtttctcaga ccaggctggg acccccaaga aagcaggggc ctggccacca ttgctctcct 8700 ggtatcctca gtgctggccc aagccctctg cacatggatg ctcaaaaata ttggtggaat 8760 gagtaaagag atgaatgagt gaatgaatga atgaacaaat gactggccag aggctgggag 8820 gtacagagca gaaaaaaaaa aaaaaacaaa aaccctaggc tggagcagat ggacctattt 8880 gagagagagg gagggagggc agccctcttc agacctggta gtagcactaa cctggctcca 8940 ccacaggcag gagcccgagg tgtggcttcc aacccagacg gctgcggtga ggatggagcc 9000 gcactgggga gcctcttaag ctgccagcca gtcagctcgt ggccaggctt gggtttcatt 9060 agagtgagac atctgttcta taaacccttc cacttagaag cttttgcctt tcctattcat 9120 tctcttgatt ttttttaaag aaaacttctt ttgaaaattt ccatccaatt ccaaacaagc 9180 taggacgaac aaaggtatgt gcctgcccct aggcttggat agaggactct agcaacttga 9240 ggctgtgggt tcttgaatct ccacagtgtg cccgacactt gcctaggctc tgaagatccc 9300 agataagtaa gacttgaatt ttgcattaac agagattaca attgggtcag aaaattacca 9360 ttgtgtgcgt taagcatgag gcaggagaca agcataggct ggttgtgggg ggacttagaa 9420 ggtgggtagg atggggcagc agtgggaagg aatgtgtctc attgtggaat cctgtgaaga 9480 tccagggaac tggggccaat ggccagttaa ccctgggaga gaagcctggc aacaccaaag 9540 cccatggcca tgccagcaaa tctcaagtgt ttattgagca tctactatgt gccagagcct 9600 ggggaaagtg ctggggatac acagatgagt atgatgaaac aatatacaga aataaacatc 9660 caatttcatt taatgacata aaaactgaga attattcatg agatgccatg ggaacctaga 9720 ggggagccct cagaaagcct gaggttcacg aagactttca gagtttgttt ttttgagaca 9780 gagtctcact ctgttaccga ggctggagtg cagtggtgtg atcttggttc actgcaacct 9840 ctgcctccca gccccccagg ttcaagcaat tctcctgact cagcctccca agtagctggg 9900 attacaggca tgtgccacca cacccggcta atttttatat ttttgtagag atggggtttc 9960 accatgttgg ccaggctggt ctcaaactcc tggcctccag tgatccgcct gccttggcct 10020 cccaaagtgc tgggattaca ggcgtgagcc actgcgcctg gcccagagtt tgttttttta 10080 aactaaatat aatatacata atagaaaagc gtacatatca taagtgtata gcttggtaca 10140 tttccataag caaatacact tgtgtaacca gcacctggac caagaaagag aacatcatca 10200 gctttccaga agccctccac ccccatgccc tctgctggtc actacccacc cccacaaaga 10260 tagcaacgat cgtgatttct aacaacatag gtaggctgtg cctgtgtttt gaaccttatc 10320 ataaatgggg cattattttg catctggctt cttttcctca ccatgtttga ggattcaccc 10380 atgttgttgc tggtggtcat agttcgttca tcctcattgt ggcatggtac ttactatggg 10440 tgaatacaac ccagtttacg cattccactg tcgatgggca accagacatg ttctagttgg 10500 gagtatcatg gctaccaccg ctatgagcag tttcgtacgg ttgtctagtg agcatatgga 10560 tgtgtgtctg ttggttaagt ccccagcaat ggaattgctg gatcagagta gatccaaggg 10620 agtctcaagg gggtgagaat gactttccta ggtggagaag tgtgggatgg gtcactggct 10680 tgggccagtg atggagggcg agcagaggga aggacaggag gaagccacag tgggaagagg 10740 ggcttgagga ccagcagcac actgaggctc tgctgattgt tccccgctta cctgggttag 10800 tttccagtgg ctgccgtgac aaagtaccgc aaactgggtg gcctaaaaca acagaagttt 10860 attctttcac agttctggag gccagaaacc caatatcaag atgtcagcag ggccacattc 10920 ctgtcaatgg ctctagggga gaacccactg cgtgcctctt ccagcttctc gtggttacca 10980 catcactcca atctctgcct ctgtcttcac ctcaccttct tctctgtgtg tgagctctgc 11040 gtgtctacac aagggcaatt gacattggat ttagagccca ctcagataat ccaggaggat 11100 ctcctcatct caagatctct tacttagcta catctgcaaa ggccgttttt tcaagtaaga 11160 tcatattcac aggttccagg gattatgagg tagacacacc tttagggagg ccaccattca 11220 tccaccacac tttccctcac tcccatgcta acacccccag ctcatttccc ccctttgcac 11280 agctctgagg caggccactt tctgatggca aatcatgtct ctcttggcca gctcaggcca 11340 agcttcctcc aggaagccct ccctgaccca caggtacttg agattcctcc agcactaatg 11400 tataaggctc tgtccagcag tatctgtggg tctgaatctc tcctcagacc gaagtttcct 11460 gagggcaggg aacacacact gttcacaggg gcacacacac tttctccagt gctcatccag 11520 ggctctgcat aaactggttc tggcaataag gaacttgggc tgaagtcacc tttccagcag 11580 tgtctggtcc agccagggca tcatgcccag taacagcccc gatgtccaac acttccccat 11640 gtcctcttct ccccctagac actgccacct taggggtcct ggctgttact tcgtcccagg 11700 gcctgactgg tagtcttagt tacacacaca acacagggga gggaggaagg agcttggggc 11760 aaagggaggg agtccagccc tttgcctttc cttggagaag gcaagggaga caggaatgcc 11820 tttggctggg agaccctggc cagagatcag aagatcaaag gatgagaaga tcaggggcga 11880 ggcaggtggc actcacttgg aggcaccaga gagaagcggc agctgctgcc tcctaagggc 11940 agggctgacc cagaacgccc tcctctaccc acagcctccc cagcctatct ttcctaactt 12000 tatagccctg cttctccacc aggaggggct ctgttggtta catgcaggtg aggagaggga 12060 tgtccactga cattcatgct acacccatgg gttctcccat ttaaacctca caacattatc 12120 ttatggggaa tattagatac tattttatag atgcagaaac tgaggcacag agaggaaagg 12180 aactagtcca atatcaaaca actgctaagt gttagggcca agatacaaac tctggcagtc 12240 tgaccccaga gccactgagg attacattat aagggcaggc agagaactct attcagtcca 12300 actgcagcat gagggagtga ggctaggtat caggaaggac ttcttgttgg gcaggtattt 12360 tagatccaga tcttggatct agtctttcta tagctctgat gaaccaaata gttcgggagt 12420 tagggaagag ggcagatggg tggggcgcta tccagaggga agggcatacg agagaagcgg 12480 cagaacgaga gaggatcagc gcggagggtg gcgggcactg gaccgcagaa ctggcactcg 12540 aggtcccagg ggccggaatg cgcttggagg gagagggccg ctcagcgagg gcgggacaga 12600 acctctcccg ggctgggagt gcacggcgcg gtgcgcccag gtaggcttgg gggaagggtg 12660 ccgagctccg gaaagttcca caggtcctcc gcagccctgg ggagcaggac ccagaaaccg 12720 acgcgacggg tggggcgcta cccgggccgg ccctaatccc cttccattgt tctgggagcc 12780 gaggccgcct ccccacttcc ggaccctttc agccatggag cgggcggcgc gggcaggggc 12840 gggcaggggc acggccaggc gggctggggt gggcaggggc gcgcgcgcga ggctggtccc 12900 cgcccccggg gccgccgctt agctcccggg tacgtgcgtg cagctagggg ctccactgaa 12960 ccacctggcg tgggttcgcg ctgccaattc ttaactttgt ggcacagtgg gaagtaactt 13020 gatatttccg aatctccgtt tcttcattta aaatgggaat aaataatgct tcgttccctg 13080 acctattaaa aaggatcaaa ttaaatgaaa ccaaggatgt tcaagagctc tataaactgc 13140 aaagacgtgg agtgggggtc tggagaggcg gaaaggtggc gaaacctctt aattttgctg 13200 aacctcatct gtaaaatggg tcagtgaaat ctttagcaca gtgtgtgatc cagcaaacgt 13260 agctcttttt attatatgct ctgaacttta tcttctgctc ttttctccta gattcaaatt 13320 gagtctggga tcctgtccaa tttagaaaac cctctggcta gggtaggggt aaatggtgtt 13380 tgggtgcacc tgggtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgcgcgcg cgcgcgttgg 13440 tgagttgagt tgctgggttg ggaaggaggg tgctctgatg taaaagaaaa gctcccatct 13500 gggatggggt tagtgagggt ggtgggggtg gtaagggtgg gactctgaaa agagggcttt 13560 ctccagtttt ccacccccag acacaccctg cctgtttatc catgaaataa aggcagcagg 13620 acaaggagct tcagctaaac aaacaaggct cagagatgtg gtctgacttc acctccccaa 13680 tccttgtgca ccctagttct tcccacctgc atcagctcca cccagttcct cttctggtgg 13740 ccctggctgg gagctcaggg atttccatgc gaggagggaa gtgcagtttt tacccatgga 13800 gaggtctgat ctaataggat atctcagtcc ttctttctaa ggggcttcca gtctgatagg 13860 gtgaacagaa ttcacgccca catacctcca cacacagaaa cagtgccctc ctagatggag 13920 ctggagatca gaatgagggc tagaagagat gctggtgagt caggggagac caggcttagc 13980 aggattgagt ggccaagtcc tctaatgcac agccactgcc gttttcatgt gcaggattca 14040 ttccatttcc ctcctgcctc agaatctgtc ttccgtccct cttcaatccc ttgtccagga 14100 cccgcaggtg ggatgcccac ccctcggaag ctgtagaaaa tgagccttga agctgcgtgt 14160 gtatgtgaca aggacagaca actgggagct gtggaggagc tcccagcctg tctggggaga 14220 ccagctacat gaatgagcag tctgagatta ggagatgaca gttttaatcg agcacaggat 14280 cgtgtggcct cactttgtga ctgaacctgc agagatcaga gtcaaaaggc caaagggatt 14340 agtctgagga agccctgtga cacttaagtc ccagtcccag cacttgtcac tcaggcttgt 14400 aattgtccac tctgttgtca ttgtccccac ttacttggct ctttgaggac aggggagcat 14460 ctgtgattgg actttgccgc ttcgtcacct agcatagtgc ctgatgtata ctaggtgctc 14520 aataaatgtt gaatagacaa atggctgcat gactaaatga atggttgaaa gttggaggct 14580 tcttgcagga ggaggtactt gaactgggct tcagtaaatg gttgagttgg gtggtgatgc 14640 tcaaaggtaa catttgttgg ttacttacca tgtgctaggc actgcctaag cactttacca 14700 gggaggaaat taagacaaag acaggtgaag taattatcca aaatcgcaga agccagtaag 14760 tggcagagct ggaaatcaaa cacaagtcag tctgacaaca gaggctgtac cattgctgcc 14820 caatagaatg ccctgcgatg atggatgtgt tctagaactg tgctgtccaa tacaggagcc 14880 actatcttta tgtggctatt gagccttgaa atgtggctag tgcagctaag gaactgcatt 14940 ttaaatttaa ttttaatgaa cttaaatata gtcacaagcg gctagtggcc actgcgctgg 15000 acagtgtagg tcttgcgttg tctctcagag aggtagtaga gagcagaggg ggcccagaag 15060 cagtcccccc atattttcat tcctctccag aggctctgct ctgtcatact cctgggctgt 15120 tctcaggtca cactgtggct ccggctgggc ctgggaggcc tgggtccctt taacttctct 15180 gcctgtgggt ggcatggctt ggcaaggagg aaggagccat tgaaggagaa gcacacacac 15240 acattatgca atcccagtgc cacgctttgc tcttgagaag cttctcctgg ctctgggcac 15300 aggcagggga acacctcccc aggctctctg agagccacaa ctaattgcat gggggcagga 15360 gtgacctttt gcacctgacc tgatgtaaca cttctgactg ggattcagga acttgtgtgg 15420 gccactccag cttcacagct gtgccgggga gagctagaga tgtcccctca gcctgcgatg 15480 ggcctaagtc cctcccagga ggattcccct gcagtgctga gggtggggac cggccccaga 15540 actgtctcca tctcctcact ctgcccaggg gcccacaccc ttggcatgct gttgctgtca 15600 gattaacccc tttcagtaag aagaggtcag agcaatacga tttaacctga agttggttcc 15660 tgtggtggtg ccaaaccatg atttcccaaa tctagcattt ttgagagaga ggaacctctc 15720 ctgggtctcg tccctccctg acctttctct gccatcaggg ttttaggagc cccacaccac 15780 atcgatgaaa caccacgatg cagtggacag tctctgactt ctgacttcca tggtctttta 15840 actcagggca aacaaaacaa aacaaaaaac aaaaaacccc acctttttgt ttctctcccc 15900 tgacttttag gaatgtcttc ccctgccttt atccctccca gccccacctg aatgaattgg 15960 gtcaccttcg ggcgacactg tgggtgccca gcatctccct ggctcagtct gacctgcaca 16020 aggtttgcct ttgcttagca actgccaggg actctctctc ccctcacaaa taacccccca 16080 ctccaggcac ccctccagct tctcttttac tatcttcccc cacatcctgt cctctccatc 16140 acaagccctc ctgccactcc aggcagtctg ggagacattc cttttaccaa caagggagcc 16200 aagaaagggc aaaataaagc agtagccctt ttatttaaaa atacagcatt gttctcacgc 16260 aggactctgt gcaaagccag agagaacagg ttgtgctctt atgtgtgtgt gaagatagac 16320 acactttttc ctgatggaag tacactccct tgtttggata tttgtgaaaa ttgcaaaaaa 16380 tctgagacaa ctttgtgtca tcttgtgtgt ttggaatctc aaagatgata gctagtattc 16440 aagtcatctc tggtaagaag gtgccatcta aagtggggag gctgtgctag aaactaagat 16500 caatctggtg ggtggaaaga gcatccccca ggattttagg agacctactt gtaacaccca 16560 ctctgccatg aacttgccat gtgtccttag gtaactttgt tcctctgagt ctgttcctgc 16620 actagcaaat tgaaggagtg taggtagagg aaagcgcctg ctattcaggg ggtgtttgga 16680 aatgcatggg gtgtttttct tcatttaaaa ttttctttta agagagaaag tctcagtctg 16740 ttgtccagcc tggagtgtag tggtactacc ctagctcact gcagcttcaa actcccaagc 16800 tcaggggatt ctcctgcctc agccttccaa gtagctggga ccctaggtgt gtgccaccgg 16860 gtctggctag tttttaattt tgtataggga tgaggtctcg caatgttgtc caggctcatg 16920 gggtgttttt gatggtcaca ataataggga gatgctccaa acacttatga gcaaggccag 16980 ggaagcaaaa agctctgcag cgagtgggac agtcccgccc agtaaagaac tgctcatcca 17040 caatgccaac ggagccccca caagtaacat gaggtgctct ctggtgcttt cctctctgaa 17100 ctcagttctg tgggttgctc acgtctcatc catgccccaa ctcattctgc ccgggaagcc 17160 ttctctgctg accctctctc ctgagtgtct gttgtactct ccagtctggg cacatggtct 17220 agaacctaat tgggagggct gcagagcagt ttgtaaacta caaagcggag tacatatatg 17280 gaggtttgcc gtccctttgt gagtaaaaag tccttcccca gcaacattgt caactccttg 17340 atggcccaga ccttgtcctg ggctttgtct ccatctcaca cagcactgag ctcagtgcac 17400 agaagcccat gggaaaacaa ctttcatgac cttcagggct ggccgggaac ttagcattcg 17460 tctaatggaa ccaggtcctc tgattccaga ttctgtgttc tttcctctct accccatcac 17520 ctgcttctct tgttgacaga aaaattcagc aagtgttctc tgagcaccaa atggtctccg 17580 tggcggccag cctgtttcct ggcttttttc agtgggagag aaccccacct ttggactctc 17640 aacagtggcc tgtcctgctc cttgccaaac cacgagctgt ctcctgccat ggcaggatat 17700 gtgcccggct cagcctggcc ctcatgggga ggggttgctg ctgaaggttg tctgggcagc 17760 ccgttaagcc ctagtcagag actgagagga gaaggctctg gagaaagctg ctggtccact 17820 gcaggagcca aaggattatt tggaggagga cctcacttta acttctctag aggatgccta 17880 attaagagaa cagggagcgc tgtgagcaac agccttcatt gattgggtgc cggcttggaa 17940 ctaagcatgg ggtagcttct ttatagaggt taggccattg tagcagctgg gggagacatt 18000 gacactaggc agtagaagaa cttccctaga gtaaaggctc agccacagga acatcacctg 18060 tgtatgagag taatgcactc atttggatat ttattgagca ccccatttgc caggcaccct 18120 gataggactg gaggatacac agatgactaa ggttaagtcc ttgccttgaa gaaacacata 18180 gtcatctgtg tcattctata ctaataatta taattcagtt agatgagtag aatattaata 18240 ataaggcata gttattgagc actgggtttt atttatttat ttattttatt tatttttttt 18300 gagacagttt cattcttgtt gcccaggctg gggtacagtg gcatgatctt gactcactgc 18360 aacctcttca tcctcccagg ttcaagcaat tctcctgcct cagcctccca agtagctgga 18420 attacaggtg accttcacca cacctggcta tttttttgta tttttagtag agacagggtt 18480 tcgccatttt ggccaggctg gtctcgaact cctgacctca ggtgatccac ccaccttggc 18540 ctctcaacgt gctgggatta caggcatgag ccaccgcgcc cggccagcac tgtgttttat 18600 atttatttac tcatatactt cacactacaa tttttttttt tgagacagaa tctcattctg 18660 tcgtcccgtt cggagtgcag tggcatgatc ttggctccct gcaacctccg cctcctgagt 18720 tcaagtgatt ctccctgcct cagcctctca agtaactggg attacaggca cctgtcacca 18780 ggcccggcta atttttgtat ttttagtaga gacgcgattt caccatgttg gccagactgg 18840 tcttgaactc ctgacctcag gtggtccacc cgcctcagcc tcccaaagtg ctgggattac 18900 aggcgtgagt caccacgccc ggcccacact acaattcttg tgaggtagct agtgtcatta 18960 tttcaatatc acaggtgaga aaatggagac ttagagatgg gaagtgacat gaccagggtc 19020 acacagctag tgagtgttgt cttgggggga taacaacggc aggtgctgtg ctacatgcat 19080 ggtaagtgct cagtaagcac tgttgagtga tcatatgcaa aatactgtgg ggacaggggg 19140 ttctgggagc acctcacaaa ggaggggact gtggggcata gccaacccca ggggctcggc 19200 tcctggccac tgtagctggc cttttcctga aagcagatct taaatactcc tatcagacac 19260 acacacacac acaaacacat acactcacac acattcacac acacacacac tatgtgaggt 19320 gatgactatc ttaattagtt tgattgtgct gattatctca caatgtatac agagattaga 19380 tcatcaagtc atatgcctta aatctataca taaataaaat aactggcctt ttcctggctg 19440 cagagatggg caacctgagt ccacagtggg gcagtggaaa aggggtttct gcccctccag 19500 tcttttctcc agcggggcag gtgggcacag ttcccccagg tccaccccag gggacccagg 19560 ggcttgtttt tcactgtttc ttcttcccat aggaaaaatt caaacagacc caatcccctc 19620 gggccctggg atatgagcca gattgcctcg gcggagggtc ttctatgacc ccagcccctt 19680 tcagctgctc tctgacccct ccatccccca gcatcctccc tcttatcaaa cctgttccct 19740 gtctccatgg tgacaccacc ttacccagtt tccttgggtg gtctggttgc ctcacagggt 19800 gaaactctga gaacattttc ctcatgtgca gccagaaact tccacgtccc atgcccacaa 19860 ctgcccctct cttctctcct ctccattccc ctcctgcacg tctctccctg agacatttcc 19920 cattcccatc ccatcatggc tgtgtccagt tgagaggact catagccctt ctgggtatcc 19980 caagggctcc caggaagaag gcttcaggat ggagttacaa cccatcatct caatcccctt 20040 gctgtacatg cataggtaca gataaacaca tgtgccctca cacatgacac acacatgccc 20100 agcaccctca aaacaagtac atccaagagc ccagctatcc catcccatga cccccacttc 20160 ttgtacatgt ctacaagttc atgcagccaa gtggatgtag ccacaaccaa accccaagca 20220 cccaaggatc aaggtgtcct cttatttccc ctgggtccag gcagggcctg aatgtttgag 20280 ctgtgccctc tgagttgcac aaaaaaccag ctgcacaacc aacctcccag gacagtcata 20340 tgtcagtagg aaccaccaac ataccaacat gacagagcaa cctctgtgtt tggagtttct 20400 gtgtgtcagt tccttccctg tcattgtgca ggcacagcct ccccacagca gggcccagtg 20460 cctcttgcca gggctcttag ccattcccag ggctggtggc attggcttgg tggctctcgg 20520 ccttgaaatc cccacttcac ctccctcttc cagctctgca cgctcctctt ccttcccctt 20580 cccctggcac cagccttacc ccttttcctt ttgtgagaga gccagccctg aatggggctc 20640 cctccacccc tgaacagttg aaattcaccc tccttgcccc accccacccc ctttactgag 20700 cactgcagct gggctttgtg ttgttttggg gcaggatgtg ggggcgggtg gaggggttca 20760 ggccccctcc tccgcagggg agaacaatga ggccgctagt cagggtggtg gggcagaggg 20820 agtgggtggg gtgggggcca tggagggaca tcagaggctc tgcggcccgg cccctggggg 20880 gacagctgag tgatgaggcc tgagtcacac gccctgagtg ctccttgaac tcccgctgcg 20940 ttcctgtcga gacacatcca gaaatgcaca gggtaaatat ttagaggtaa tcattttttt 21000 tcggcacctc gagcctcctg cctcactgag aatgagccag gggtgggaca ccagggggag 21060 gagtccgatc tggcccagct tctgcgcttg ggtttctgtg ggtgccaccc tacgttcccc 21120 tgacactgtc cagtgtgctc ctgcaccttc gcatttcagc cacaatccaa gactagtggg 21180 gcctgaaaac atcctgagga ccaaaccctg gcttaaaatc catccacagg ctgggcgcag 21240 tggctctctc ctgtaatccc agcactttgg gaggccaagg caggaggatc acttgaggtc 21300 aggagttcga gaccagcctg gccaacatca tgaaccccat ttctactaaa aatacaaaaa 21360 tagccaggcg tggtggcagg tgcccataat cccagctact caggaggctg aggcaggaga 21420 atcacttgag ccccggaggc gaaggttgca gtgagccgag atcacaccac tgtactccag 21480 cctgggtgac aacagtgaaa ctctggggaa aaaaaaatcc atcaacaaac tctctgggtt 21540 ctgcctctct cccacctgaa actatggcca cagcacacca cctacagctt ctgcggatcc 21600 catttctaga ccggcaagcc cagaggtgtt ctgcagggga gcagggaggg aatggtagat 21660 gagaagcagc gctgggtgca gcatgccagt tccttggttc taccctgcac gggaatgtct 21720 tccttccttg gaggacatgg agtccagagc ccaggcttta tcaaagataa aacatttgag 21780 actaaacagt ctttttatag attggaacag gactcccaag cacacgctct ccaggctgtt 21840 cccagcctgc ctttctgggc acaaagaaag cacacgcatt tcttgggggg aaatgcagac 21900 actgaggttt gagggcaaca gtgcatgtct agggaaagga gaaagcctca agaaggcttg 21960 ggggcagtgc ccagcttgtg gtggtctggt ctgagcccac ctctgtaccc cctcagggaa 22020 ggggcaggca gagacgagaa tcaggcatta ggagtttgaa gtaggatgga atgcaaatgt 22080 gtcaggcaca cacacacagt ctcacacact gttgctcatc caggcacaaa ctgtgaagaa 22140 gatgccaaca cagggtagga cccacagagg aaggtaccaa ctcctctcta ctactttcct 22200 ccttccatct ttccctcagt gtcctgtggc aagtgtccca gccccacagg gctgtggaaa 22260 aggcacccgc ccttgctcca acngccttnt nganatncac actcgcccac aggaccccga 22320 gggaaaggca agaggagaaa agcaaatatg taccttgtcc atactattta ttaggcagtc 22380 tgtaagcatt agctcactta atctttacaa gcaccctacc agcttggtgc tgccctattt 22440 ttatagatga ggtcaactag gggcccagag gtcaagggcc caacgtctga taggtaactg 22500 cagaggagag atgtgacctc agggcctttt gacttcgaag ctcagtgctc ctttctctct 22560 gttttcttga gtgtaaaatg gagataatga tggaagctaa tttggggtta ttgtgagaat 22620 ggaaggaggt tgtttgggta aagattagcc ggtgtctgac acagagtgga cctgcagtaa 22680 atgccagggt aattattgtg attattttgt gtgcagggcc tctaagagga cagtgacact 22740 gaaaccaccc acagggtctg tcagataata tgagcttaag atgattgagg ctgtggatcc 22800 gcagagggac tgggatcctt accccacctg ggaatctagt ggtgtgccag ttctgtgatg 22860 tgccaaggtg tcatgtcttc caggtggact aagacagtgg tccctaacct atttggcacc 22920 agggaccagt ttcacggaag ataatttttt tccacggaca ggggtggagt gtgggggatg 22980 gtttcgggat gaaactgttc cacctcagat catcaggcat tagttagagt ctcataagga 23040 gcatgcaacc tagacccctc gcatgcgcag ttcacagtag ggttcacact cctatgagaa 23100 tcttttgctg cctcggctga tctgacagga cgtggagctc aggcgggagt gctcaccagc 23160 ccgctcactt ccctctctat gttccgcctg gttttggtct gctgcccccg ggttggggac 23220 ccctggacta agggaagcaa acaggtagag cctggtagac atctcaaagg gcagtgaaat 23280 gccactagct ctgggattga atcagaggat ggtcccccaa ggggagccca gggacagtcg 23340 taccagttca tgaatactga atgagcctgc tccatagatt gaaatagcag tgatctctgg 23400 gcctcagttt ccccactagt aaaaagagtg ggtgagatat tatttgtatt tggcgatcct 23460 tcccttcctg ctctgaaatt ctatgaattt tactctagtt cctcgcttta tagggaggcc 23520 agtttccttc ataatcccaa tcacagccag tcccagggga gtctcacaaa ggacaggtga 23580 aaggcagagg ggaaagtggg agaccatata gagagaatca ccaaatgacc cgggaggcct 23640 gactcccagc tcagttcttg ctcttagaga gaacgcagct gctgttgtca gactggcttt 23700 ggagtcagaa tggcctggtg gcgggtactg tgggagcctc ggaggtccct cgaggggggt 23760 tggcacctgg acagtgccag atggcccaag tggggagttc acagctcctt tttccatctt 23820 ctctccacca gggacctcag catgaacaac ctcacagagc ttcagcctgg cctcttccac 23880 cacctgcgct tcttggagga gctgtgagta gatgctttgc agggtgggag gcaagcatgg 23940 gctctgtcta atatctctgg aaccagagct ccccaggagg tggggtggag agaagagaaa 24000 ggggcttggg aagctaagac agttcttgac agcagggtgt tcttcagctg acacctgccc 24060 caggtgtgaa ttgacatgct gggccacatg gcatgggaag tctaggatct agctttcatt 24120 cctctttgaa accagctcct ctttgcagca ttccctccta acttctccct gggagtcatc 24180 tttgacctgc tgtcatcccc tgtgtccagc caccagcccc tccccaagtc ctggtggttc 24240 ttctctggga tgtctctcac ctccaccctg cttctctagt cccactgcta ctgccccaga 24300 tgtcccctca catctagggg ttgaaccagc ctcctaactg acagatgccc tccttccatt 24360 ctaaattact ctgcacacct tcaggatgca tctcaccagc tcaaaagcct tctgtaattc 24420 cccatggcct aaaggatcca tttcttagac cctcccttac agttaaggcc catttcctcc 24480 acaaagtact ccctggtgaa tccctgccca cagtggtcac tctcaggttg ggattcttca 24540 atctggcctt atatgctagg ggtgctcaaa agtaaatcat ctcctggatt agagtgactg 24600 gactgcctgc tctgggaagc caggtcattc tgtcccaatc cttgctttca tcctagctct 24660 gtatttccaa cacctcccag ccctgttttt tctaattttc acaccacata gatccttgcg 24720 gctctagaaa ttttgcttaa acttctcaaa ctgagtgtta cagctctttt agaatttgtc 24780 tagctctttt agaatttaaa taaaaaataa taaacaaaga aacttcgcaa actgtgagtt 24840 cctggccccg aaagcattca caatttcacc tctaccctgg ccccactact cccctaacag 24900 accttgccac cgcgacccta gacagtgctc cccagggatg cggtcaacct gcccctgttg 24960 gagttttgac ccccagggcc atggcttggt atccagacca agtacttgtt gaaccccctc 25020 tggtagacag gcaagctcaa ggtgctctgg gagaaaacaa gataaaagcc ttcctgccac 25080 ccccagggaa tgtccaggtg aggctgaaga gtagtgtggc cagcaagtca gcaggggagt 25140 gtggcagctg ttggctcagg cacccagggc attctgtgta agggagtggt cagggttggc 25200 ctggccagta ggcaggaaag ccttcctgga ggaagaagat taaaccagtt ttcaaaggag 25260 acggacaggg taagaggcag tggaggggcc ttttgaaggg gagatgaccc agcttagatg 25320 ggaatgcaaa gcaagagcaa aggccaagga gctcaggcag gtgacattgg acacgacctg 25380 agaacttcag gctgactttc cgtctcccac gacctgagag gtctggaccc attttagagg 25440 ctccacctaa aggacctttc cacggcaaat acccacctac agaacccaag ctaggagacc 25500 tcagatgtca cgatgccttt ccacatagtt tttttttaaa caataaatgt caaataaagg 25560 gacttttatt agagatatat gttttaaaaa tagtgtctga ggtatgccag cacaatctcc 25620 acatctacaa gcctcttggt aggaactgat gcacaaaata atgtccctgg ggactttgga 25680 gatcaggcag tgacttctga atcatccacc attaaattct gctcagtcag gtttcagcat 25740 tgagtgtatt tcgttgcatt tgctcctttt agaattggtg attgccccga taaacatgga 25800 aacgcttaat tagaccagaa tgttagatga attacaccac cccatttcct taaccctctt 25860 ggattagtga gggttagctg tgtagtttag ggcctggaga gacacagaca caccccctgg 25920 tactcctccc tctactgatg acaccatatg atgcagtcca gcagttgaga gcaggcggtg 25980 ggtaatccag gttagcacag gagaggatgt ggggaaactg tggccgtgag ccttcaaatt 26040 agtcaggtct gggtgagaac accaggctct gccatggact ggttctgtga cccctagggg 26100 acgtggttga cttctcctag cctcagtttc ctcatctgaa atttggggaa aataataggc 26160 actttacagg attattgtga gaactgaggc aatgcatgtg aagtattaaa cacatcgtag 26220 atttcaataa atagtagttt ttaccaccgc cctcccttcc cgtatctcaa gacactttct 26280 tatggaggtc accacctcct tgggttacct gccaacatcg ctgacccttg tctctgattt 26340 ccaggcgtct ctctgggaac catctctcac acatcccagg acaagcattc tctggtctct 26400 acagcctgaa aatcctgtaa gtataggtac acctcatttt acatagagct gcagcactga 26460 gaatggtgag caaatcaaag ttttagaacc caagtcttgg tttaccactg acttgcttat 26520 tatttcctta tcttcacttt cagtttcaat tgttatttcc atttctttgc ttcaccttgt 26580 ctgtaacaaa ctttatgatt tgtaaataaa catttaattg tacctgctac attcagccac 26640 actaaaagga agtgaaacat ttaaaaaaat taaagtgtat aatgaccatc gacataggtc 26700 tttgtgggac tgagttcagt tacagaccca caggtctctc cccatgacct ctagcccaag 26760 agacggataa gactgtttct ggagagggga aatacaggtg gcttttcttt ggcttttctt 26820 ccccttgcaa cccaaccttt gtctctgtgg ctgcagtact gagtttccat tctggctata 26880 aacatattcc ctccttcggt cttcatgttg cttcctccct gctcctcctt tacctattcc 26940 ctttgaagca aaaggaaagt gagagttgga gaggaccttt tgttattcca cctagcttcc 27000 acttaccatc aaactgatgg cccatttcag agaaaggaag gctgagacac aatctcctca 27060 actcatgact caccttgttc caactaagac tgtcctccca aagcctcctc atatgcttat 27120 gatgagtgag ggcaagagga attgggagga agcggatgca tagggactga aggtagatag 27180 gagaggttcc cagggaggtc gcgttgccac agggtctggt ttgggaagaa aaggaggata 27240 ttagggactg atgctggagc aacagaatga gcatttgggg cttccctaaa gtggtgagtc 27300 attcccaact ctgctactcc cttacccctg acttgcaggt ctcctttgta cagttgagca 27360 ggttgttcat tgcaccagag catctggtta aggaagtgag aaggggctga atcccgtcct 27420 gtgccctggc ttggggctgc attcacccag aaagcgatat cttttaaaaa tgtactcaga 27480 ggtgctgatg ggctagtggt ggtcctgccc gccttggccc atgagttcca ggcttccagc 27540 tggtcaagtt caacactcca catctaagca ctttctcctt tccctgtgtt gttcgtctct 27600 ggcccctctg cctgtgaatc tctaactccc agggctctct ggtctctatc tttcccttac 27660 tctctgctct gtccttgggt ctctcctgtt gtcaccttgt ctcccccttc tccttctccc 27720 tgccccacta ttccctgcct ctcagttctg cttcttcctg tctcctctct gtgtctctct 27780 ccccataaac ttctctgccc tggggagcag gggaggcatt ctgaaacaga cacacagtgc 27840 aggcggccta cttggtggtg tgtctgtgcc cagtggcagg ctcagacctg cagcccggat 27900 tagagagcag ggctctgttt actgggcctt cagaagatgt aagactcacg tcccattcct 27960 aagactccgg cttagagatg agcctcctct ttcttctttg cctgcattag gcaataaatc 28020 aaagcttcag agaaaccaca ggcctcccgc ccccctttct gcaaatggca cccttggctc 28080 agtgagtagg gcagcagggg gaggttgtgg gcagaagttg agtgggagct gaggacagcc 28140 aggctgtctg ctccccactt tgccattgag cctcattttg ccccatcctc aaagatcacc 28200 tggtccccca aagcctttgt gtcagtcctt ggcctacaga acttctctac cttagggatg 28260 ggttgggtgg tcttggggaa ttatcctctt acatgcttca aagcagaaga ttccacctgc 28320 atcctaggca cgaattactt tctgatgtct gatgtcaatt tccctcgtat ttccttccat 28380 tgcttgcttt tcagagctgc aagaaaaata gagcagtagc accatgataa tgacaaccta 28440 tttttgtacc aggagacagt caccttagct actccccagt cttctctggg cctgggaccc 28500 ttatctgagt ggcctcttct tatttcagag gctgggctct ccctgtggga cactctgcct 28560 tggtccttct ctcccgaggc aaaggattaa ataaagagga ttccctctct gattctttct 28620 taaatgacta tggcagaagc cctctctcat ggtctttccc acaccccccc acacccccac 28680 ccatccctgc ccgatctgga gatattactc ttaagatctt cttcttctga gagttctcta 28740 agtcttcaac ccacaggctc tggccaggct cagatcaagg gagtcagcta ctgcagggaa 28800 ctttcagtct cacatcgcta tctctgtcct gtagagctgg caaggcccag acgctagtct 28860 taaccatcac tagtctcacc atcccggctc agcactggag ctgtgctaag ggaagccatg 28920 gctccctctg ccctaggcat gtgggaaaat gcccgagcct ctgagggctc ctgtcctgcc 28980 tgtttttaca caggggcagc tttggccaga gctcctgcat cttgacccgt ggactaaggg 29040 tgtggggatg catgtgtgcg tgtgtgtgtc aggcttctcc agtctttatc tcaaaagttt 29100 cctgtctcgt ctccatcaca ggtttctttc cccagagctg gcctcccaga gagctgggtg 29160 aggcctgagg cttggtcatc ttctcacatc tgcagaccat ctcctccctc cttatgccag 29220 attggcctga cataagatgg tctggcatct agaaagactc ctgctgggct tcagttgctt 29280 cttaccaggc ccagatctgg gaaactgggg taacagtctg aggacctgcc tcccgtcctg 29340 ccctcaagag actgggcagg agattttccc aggtgccttg ctcctggatt gagtcccaaa 29400 tccttgtaag tctgtgactg tggattctta gcccatgcca tcttgataaa gactaaggct 29460 cttagaaggc agctcctgct tcctgagctg cggcccatcc catatgaaac cctggctcca 29520 ggccacagag gagtggctgg gagttagcat ctccgggacc catcccggag gaaatctccc 29580 tgagccaacc tctagtgtag cccttttcac cctgtatgga gccatcttca gttgacatta 29640 gctgtgttta ctctgtgcct ggctaggcag tgaccatgcc tggctgagcc tgggctccat 29700 tagtgtcgat gaactgaatt gaactgaccc acgaagggaa atgctggtca tttgggagtg 29760 ctgggctctg tccgagcctc accaacctgg ctcgggggag aaggctgccc tctctgagtc 29820 acccctgtac atgaggcaag gcctgctgcc ctcatccttt caagtgatgg gatgacaggg 29880 agtggatcta gctctgtgga tctcatgtgc agtcagaagc cttgaggtca gactcagagc 29940 ctcaccagac cccagaacag atgttaagga ggatctcaga tttcctctct ctgagcctcc 30000 gttttctcac cttttaaagt ggaggagtca gaataaaatt tccaacattt ttagctatgg 30060 aaacttaaaa cagattcttt gtagttcata cacatgatga tcggtgttca cactcatacg 30120 tgtgatgtgc cacccttgga ccttgttacg acatcagcac attacccctc tacatgaaat 30180 ttttttttta aaggaaactt aaaatcaaaa gcattttgtg ggctgggtgc ggtggctcat 30240 gcctgtaatc ccagcactct gggaggccaa ggcaggtgga taacctgagg tcagaagttc 30300 gagaccagtc tggccaactt ggtgaaagcc catctccact aaaaatacaa aattagcagg 30360 gcgtggtggc gcacacctgt aatcccagct acttaggagg ctgaggcagg agaatcactt 30420 gagcccggga ggtggaggtt gcagtgagca acctccttga gagttgcgcc attgcactcc 30480 agcctggaca acaagagcaa aacgccgtct caaaaaaaaa aaaaagcatt ttgtgcagac 30540 ctcccaatac ataaaacaga tgaatgaggt ctctggttaa agtgagtgaa gagggtggta 30600 cactccaggc tgcatcttac ctacccaccc cataaccccc aagcacttct acagaactaa 30660 aggagccttt ggaaaccaca ggtggatcac ctcttttata ctttttggat gtgatgtgac 30720 actcatggtt cctgccccca aatgcctctc tggccagaga gatgcttcac atgtgtgagg 30780 ggaggagtgt tgaggactag acttctactg gaccaacggc tgcatgttag agtcagctgg 30840 gagcttgcaa aactactgct gcctcatatt ccttccccag agactttgcc ccagagattt 30900 aactgggctt cagtgtgacc tgggcattgg gattttaaaa atcatcccag aggtcaggtg 30960 cagtggttca tgcctgtaat cccagggctg tggagggctg aggcaggagg attgcttgaa 31020 cccaggagtt tgagaccagc ttgggcaaca aatcaagaca ccatctctac aaaaaaatgt 31080 taaaaccttg tctgttgatt ctgatgtata accagggcta agaaccgctg gattagatag 31140 actgcctctc catatagaac cagggctaga aaaagaagta ggaaaatatc gtcacaaaag 31200 cattggcccg agagacagga cttgagtcct agtcctgatt ctgccattca tttgctttgt 31260 aacttagggc aagttacctt ccctctctga gccttcacct cctcatcttg gaaaacaaag 31320 ggctgtttcc ttccaacctt tataacccag ttgtagttca ataaaagaat gatggctagg 31380 aaatgagaac agggacatca agggaaatgg gtcagggcca aagccgactg ccatgctgac 31440 ctccagcttg atgggtgttg attccatgaa taaaaggact ttctgataat caggctgtct 31500 ggcataggat gagttgtctc ggggagacaa tgagttccct gacactagaa ggaaaattaa 31560 gtgctgtgag aagatgttac agtgtctgta ttgggaggag gtacaattca gaaaccttaa 31620 agatctttct agttatctct ctgggatttg acaaaggaaa ggatggagga atcttaggag 31680 gtgatgtagg taaagatgca ttgaggggcc ccttggagct cgggactcac ttgtgcagtg 31740 accagatgta gcctcctggg cccctggagg gatggggcct gtgaagaggc atgagcagcc 31800 tgcgaccccc tgttttctca gccatcgttt ctcttgtatc cttgctccaa gcaccctcag 31860 gcttctgcat gagaatcaag gcaggcctct gagatgggaa gcgaaggccc accctttaga 31920 tgaaagaagt caggatggct gggagggtag aaacactaag cagggcagag ggcaggaaat 31980 gtcctggatg gaatgttacc actgagtgag gctttatgca ccttctggca gcctcttcct 32040 ttgaaagatg aaggaatcaa ggtccagaga ggggagagga agtgacttac tcaagtcatc 32100 cagtaagtgg taatactgaa acgagatact aataatgata attgatagta tcagtgaatg 32160 tgctattgaa cttgtcctct gtgtgagaca ctgccattga gggctttaca gcggtgattt 32220 tatttgatcc acacaatcag tgaatgaggt cagtatcatc cccaacttat agataagaaa 32280 agagaaaact aaggcccgcg gttcttaagt agcttgcaca ctaccccaca gctggaaagc 32340 aacagagcta ggatgtggat cagggctgtc tgacttcaga gtctgcatct gtgacaacta 32400 tactgtaagg aagcccaaga gacacaggtt taaattccaa ccctgaagca aaactctgta 32460 actggaaagg cccctctggc attgggtgga gcctggattg aaggggacac tggaggccaa 32520 cagaccttca ggaacacaga gtttgcatgg aaaggggctg agccctccag ctgctcacag 32580 ccctgggctc tgccccttcc tgcagccagg cccttccctt gtatggccct tgccccttcc 32640 ccaccccttg gggtggcaaa gaggaaccta atcccagggg tccccaaagt ctgtctttgt 32700 tctttgttct cccttctgta gccttgcctc tatgaagggc tcctcctatc cccacccatt 32760 cccacagtga gatcagcatt cttttgttct tgtgatgctg aggttgggtt catatttgag 32820 attttcacat acccgtaaga ggagtttaag agagatgaca tgtgactctg accaggacgt 32880 cacataggag gcatggaaaa caactcaggc tcatcatctt actgctcagc attttctggc 32940 cactgctgta tcccaccact atgatagatg ctgtggtgta atggttaagt gtctagactg 33000 tggacccaaa ctctctaggt tcaaagccta gctctgtcag ttagcagttg tgtgcaccct 33060 cagcatgagg atgataataa tccctatctg ttggagttgt aacaagggtg agtttgaatt 33120 aatacaagtt ctaagaccag gacctggcac attagccatt cctattatta aactaaagga 33180 ccagaaattg ggctactctt cctgaagcgt ggaattacct taatgattgt gcaccaaggc 33240 ttccccctct gaaagatgga tggatacacc ataaaggggt aattgagtct cctagggtag 33300 ggctcactca ctgtgcccag ggcagtcagc aacttcctcc cttcttgctt ccccagctct 33360 tccatgaact ccctgtgtgc tctccttctc atttctggat ctaaaggcct ggagggtttt 33420 cctcctatgt ctttacccag ttccttctct tgctcagggc tctgtgcctc aggggactcc 33480 ctgcgctttc gagtgctatc ctcaaaactt tctaagtccc ctctgatgcc tggggttggc 33540 tagagtcagc agtgccacct ggagtggcgc cccaggccct gtttctcccc ttgtacatgc 33600 tctctgaccc tctaccctac acatggggtt cccaggtgcc ctgaggagct ccaggactcc 33660 tgcctatgga gtcatcgtgg ggttttggga cttaggtcct ggtccccaga ggacagctag 33720 tgagcagatc gttttcattg gctggcacag cacttctttg agggattagg tgagattggg 33780 ccatgagaat agtgcagata cactcctctg gagtacttcg aattgttcct acagacaaag 33840 gctgaaaaaa atgtatttgc ctggggcact gacaccctaa gggcagcctt atcctcatcc 33900 acatcctccc cttccataaa gaaaccttta gcctttcaga tgaatagaca cggaaactga 33960 caacctacat tttcccacat ccttcctgac caaactttaa caaaatccca agtactcttg 34020 acttcagaat gtcttctctc cttcctctcc atggtcttct tcacccctgc ctcctcctag 34080 ggtcccttct gagatgctct ccctgaacac tgggcctccc tgccccatgt acaacacccc 34140 agggacctca gcagcctctg aatctgcact aacaaagcag gggagctgga ccagactctc 34200 tctggggctc tgaggctggg gtgagaagga ggggtctggc ttgggactgg gggttcaagg 34260 gaggagtcag caccctccag ccagctctgc ataccatgtg cgtccttaaa gccctttctc 34320 ttctcccgta ggatgctgca gaacaatcag ctgggaggaa tccccgcaga ggcgctgtgg 34380 gagctgccga gcctgcagtc gctgtgagtc attagagggc tggtctggga gtcaccagct 34440 tcctggggcc tgagtcacag cccagggccc cctgcctcag ctttcccttc cctctgcagg 34500 cgccccctct cccagtccac gttccagaca gcatgccgct cctggctttg gtttctccgg 34560 ggttttctgt tgtcatcagt gtgtggctgt gtacactcct atgcatattt caggaaaaga 34620 gtgctcccac gccccactgg gggactgtgg gaatgggacc tccctaggtg aggccaccag 34680 gccagcggtc ctggggcgac tggtgttctg ccattggagc ctgggtttag gggtggagac 34740 tgccgcccaa aagaagcatc aggaaggggt tgtctcctct acctagcctg ctcttgcccc 34800 tgggctctgc tgtgggtggg aaggaggcta ggctcagata ctcattttct ctacttgggg 34860 gtgggactgt ccacagggac tcacaggcca cccataacag atccctccct ccaaggagcc 34920 ctgtagttcc cttcttgtcc agcaggggca tcgtggaagc cagacccagc ctgagggtgg 34980 aagcgggagc aggtgctcca gggtgatgta acccagccca gtgggagccg ctgggcttca 35040 tccacataat gcacagccca aggcctggaa cacagtaggt gcctcataca tgcttgatga 35100 acctaactta attatcaggt acaacttcat ccgatgggag agtgaacaga tattttggga 35160 gaagtcaaag ttggggggca agattggggc acagcagtta gctagtgggg gaaactgagt 35220 catagtgggc atgcagtgtg tccgaacaaa ggcctctcat attctcagac tttggcgctg 35280 ttcagtctgt ggatgtggaa gtcagacaac ttctgacttc tccttgggtg gaaggagaaa 35340 tgaggtggga aaaggtgact ttccttctca tgactgatat ttaggcccca ttccacctta 35400 cacagagggc cagctgtggg ggtcctggct agcagctgtg ccacaccccc cgtggtgagg 35460 gctttcctct cctactcctt ccctgggtgc cctctaccct cctgcctctg tgtctggtcc 35520 ctggcacccc tcagctgctc gatcgatcgg ccccagtggc agaacctgcc tccctgcact 35580 ctctcagctt ctgccccccc ttcccaatga gtgctgggcg tctggcccca gccctctcct 35640 ctccctccat ccagcctcac ccctggacag ggccaggcaa cccagtgtgc ccctagaggc 35700 caacgtcgga agaccagagg ccaggcaagc ctgtgcttgc tccccacccg caggggcaga 35760 ctaaacattt gcctagggca ccagcgcagc agggacagcc aaaaataagg gaggaggagt 35820 gaggagaatt tgacatttca tccacagcct caaagaggac ttcatagggg aaaaaaatta 35880 gactttcact ggacttcttc acacttttaa ggttgttaat taagtttact tttttaaaaa 35940 aaatcgtgaa tcacatattc tgagttcttg gggcttctaa aggtcttgct ttcggactag 36000 gcatggagct tttgggcaat gcagacaatt ttccctacac tttctgggaa gtgggcagcc 36060 cttgtggctg ggagaaccag accattagct tccactttca gccccattat tctcctttgc 36120 ctgggtataa cagggcaggg gacagaggtc aagtccccca ctcccccagt tataaggcag 36180 atgagaagat tttgcttttt gcaatcctgc aagaagagca ctggcctgtg tgacagaaaa 36240 tgtggcttca agtcctggct ctgatcgtga ctactgtgtc atttgccctc tctgcacctc 36300 agtttctcta tcttcaaggt ggggataatt atcttccctg cctcacaggg tgttgggaag 36360 atcaaatata tagtcaatgt gagggggatt tgcaaaaaaa aaaaaaaaga cttcacacag 36420 ctataatgta tgatgatgtt attgtgagat cgttcatcta accagtcatt gatcctccgc 36480 cgtttttttg ttttgttttg tttttgtttt tgtttttgtt ttttgagaaa gggtctggct 36540 ctgtcaccca ggctggagtg cagtgacctg atcgcaactc actgcaacct ccacctccta 36600 ggctcaagcc acccctcacc ttagcctcct gagtagctgg gactataggt gtgcactacc 36660 acgccaacta atttttgtat ttttttgtag agatggggtt ttggttttgc catgttgtcc 36720 aggctggtct cgaactcctg agccaaagca atccacctac cttggtctcc cctcccacag 36780 ttctgggatt acaggagtaa gccactgtgc ccagcctgat cctttttttt tttttttttt 36840 ttttttttaa gacagagttt ttttgctctg tcgcccaggc tggagtgcag tggtgcgatc 36900 tcagctcact gcaacctccg cctcctgggt tcaagagatt ctcctgcctc agcctcccaa 36960 gtagctggga ctacaggcgt gggctaccac gcccgtctaa tttttatatt ttcagtagag 37020 acgggggttt caccatattg gccaggctgg tctcgaactc ctgacctcgt gattcacctg 37080 cgtcggcctc ccaaagtgct gggattacag gcgtgagcca tggggcccag ctctattttt 37140 ttttttttta aacaaaattt tttgaggtgg cagaaccaga gttgagcctt gaaagactag 37200 tatgaatcag gtgaagaggc ggggaataac gtttcagaca gagggaacag cacacgctgc 37260 aaaggcacaa agttaagaag gaacctgaaa ggttcttgta tcggttggaa tcccgagagc 37320 tccccaacag ctaacacgaa gcggtgcgtt ttaacaagag cctgggtgcc ggcgggctga 37380 ggcgtaaaat ggcgtcagcc cccaaaatgg cgtcagcccc aagtgaggac ggggcagggg 37440 ttttattgtc tcctataaac agggggcgtc tcggtctgac gtaactgcta cgcggtaccc 37500 ggatggcctc tttctccatc ttcaggggta ggtgtcttcc aaccagggta ggtgtcttcc 37560 ggccggctct tttcctgctt ctgctgtctt gctggcgtac gcaagtagcc ctgcgcctgg 37620 cgactgggcc tgagaaggga ggggttactc atcccttcaa gctttcaggc cccggggaca 37680 atctttcaga accaagtaaa ttctgaagaa tttggaagga agtgagtttg gctggctgaa 37740 cttgtgtgtg tgtggtgtgt gtgtgtgtgt gtgtgtgtgt gtgtggtgtg ttgggggact 37800 ggtgggaagg gcagtagtgg ggaaagatct ggagttagac gtggtcatac ctgaaaagta 37860 gggaggacct tgctaaggat ttggacttta ttcttaagac agcggggagc cacgggaggg 37920 cttttagcag gaaagcaagg tgatagggtt tgtgtctttg aaaagttact ttggcagcag 37980 ttataggggg cagtctgaag gtggggaagt cagtgaagag gctcctaaac caaggcagca 38040 tgttagtggc caaaggaaag acagacgggg gcattggtga aaggagaagc agacaaacta 38100 gaaatatcta gaagtcatgc cagtgagact ctggagtcgg attgtttggg ttcacctctc 38160 agcttcttca cttactcact tactgcctat aagatcctgg gcagactact taccctctgc 38220 gtttccattt ctttatcagt aaaatggaag cgataatagt atctgtgtat ctcaacagca 38280 tagaatgaaa tgatttaata tacataaaag aattagaaca gcaccttttg ttgctacttc 38340 tacaatgatc attagtagta gtaatgctga ctggatgtgg gagctgaggg agaggagaag 38400 ttgagctgac acccagttct tgggctttga catcagggta taccgtcagc aagagtctgg 38460 cacgtggtaa gagcttatta agaacatgca ggctgggcgc agtggctcac gcctgtaatc 38520 ccagcccttt gggaggctga ggcaggtgga tcacctgagc tcaggagttc aagaccagcc 38580 tggcaaacat ggcaaaaccc cgtctctact aaaaattcaa aaaattagcc aggtgtggtg 38640 gtgggtgcct gtaatcccag ctacttgaga ggctgaggca cgaaaattgc ttgaacctgg 38700 gaggcagagg ttgcagtgag ttgagattgc accactgtac tccagtctgg gcaacagagc 38760 aagactccat ctcagaaaaa aaaaaaaaaa cgacatgaaa gaacgaatga acagcacaag 38820 gtgagtgctc tggggagggt gcagggaaag gggatgtgct gaggcccctc tgggacatgt 38880 tgagtttgtg gtgcctgcag gcaggataac caagtatcct agagaacctg ggaagttggt 38940 cagttgacct gcaggacatc agtgtgtgtg tgtgttggtg ggaagagggg aggtctgtaa 39000 tccatctcaa agattctgta tgtaggtgtt atgttctaag tgtttggaaa ttgggagatt 39060 cagggagaga ccgcttcagg ccccacagca aggtgctact ctatcttatt caggtccttc 39120 tgtcttagat agtgacagta aaagcaggcc caggtggcta ggctgaaagg agacagtggg 39180 gaaaaatgac aattttttgg acttccctgt gtgccagcag ggacccatca ttccatacat 39240 ctgaccttcc tccaagggac tattgtgccc ccagactaat gcccaaggcc aggctaaagc 39300 tcccctaaag ctacatgaga tgttctacgt atattgtctg attattcctc acagaaaccc 39360 ctctctaata atcaccgtct ctgttatcag atgaagaaat aggggcagac aagttcaggg 39420 cctcttccaa ggtcagagct ggtgagtggg catggcaggc ctacacccag ttgttggact 39480 ccaaaggcta caagggaagc cgccttttga tggctttcac tgggtgaccc tccctgcttc 39540 ccaaggcgta ccagcaaatg gaaatgtctt tcctgttgga aatcaacgga tcagggaagt 39600 atgtagagga tgcaccccct gcccatgccc ctcctggggt gatgtgttcc cagagcagac 39660 caagccactt aggaacctga gcaaactctt gtggacattc ctggagcctc agggaaggtt 39720 cagtcttgtg gtccactttc taccagctag aaccagatgg gggcacagta agaggtaggt 39780 ttgtgtgagt gaggagtggg gctagggcta gggaatgggg gccagccagg cattctaggc 39840 ccagggaccc agctgaatgt ggtacacaaa ctgtttggct tggcacctgg gcaggcagca 39900 cccagctcca caacaccctc ctccttggat acacttgctt cctccctgac cagtttggga 39960 ggggggagaa tggccatgcc agacatgtca tgccaggctt gagccaaggc caagagaaac 40020 cacaggaagc cagcagagcc tgggggaaaa tagaaactgg gtgggggtga ggcagggcaa 40080 gggggaaaca gcatggcaaa gcagaagaag tgggggctgc gctggtctgg gtcctgggga 40140 gctccggtgg gctggtccca ggagaagagt tggaaaagca cagtggcaag actaaagggg 40200 agaaaacagc caaacagccc aaacctggat actctagggg ggaaatgcat aatgcgaagg 40260 acagccacat gaaactagca aacattgtct agagccgggg aaatgaatca agagcagtac 40320 cagactccaa actgaaaccc aaggcagaac taccccaacc ctctcagccc aagccagcca 40380 atccaaaaca gattggttgc ctgctgagag gaaaaaggtg gagaagccag ggtggataga 40440 tgatgtgctg gtgtgctcgt gggccgggga aggcagctgg gctcaggtga cgtgggaagg 40500 aggagtggct ccctcttagc acatttcaat atgctaagca cattcatata tctcatttaa 40560 tccccaaagc cctgtaaaat agtgtctgtt attcccatgt tatagatgag gcaactaaaa 40620 cccagaggag ttaagcaact tgcctgaggt cacacagcta ggatgtaaca gagctgggac 40680 tttagtttag ctccaactcc aaagtaccag gttcttaacc cctatccttt agtacatccc 40740 ctaaaactat aatctttagg atccagagac ttgggttcaa gtcctagctc tatcactagc 40800 tacgaggcca tggcaagtca cttcctctct ctgagcccaa ggttctctgt ctctaaaatg 40860 agagagtcca gtgggatgat tggcagacag atgtcccgag tgaatatcat gcatagaggt 40920 acaggaactt cctaatgggg ttactggctc tgctcttgaa ttgtttatgc ttactgggtg 40980 cttgccttct ctgggtctca gtttccctta ctgtgcaatg gaaagtatct tttttgaacc 41040 cttccagtct cctgggaatg ctgtgagttg agatgaggag gaaagacctg agctgctcca 41100 ggagaaaatg gctgaaccat tttgtccagc attggaggtc ttaggaggga ttggcagtgt 41160 tcccagggag aaccaaaagc aagggacctc tggacaagcc cccctcatct catgcctctg 41220 atcgccctcc aaaggactat tgtaccccca gactaatgcc caagggcagg cgaacaacag 41280 gaaccaacac ttggctagca ctttccatgt gcaagtcatt gcacttattc tctcacttaa 41340 ccctcacagt actcttctgg gatggctact gctagatgtc ttgtaatccc cattttatag 41400 acgagcaaac tgaggcacag agccataaag tgacttatag cagagcctgg gtcagaagtc 41460 aggcagtctg gcccagagtc cacgcattta accagggtgc tgggctgcct ctcaagagca 41520 attctgagag ggctgaatga gatgacaagc cttgggaagt ggcccatcag tggggctcag 41580 gaaccatcgt atttccaggg tacctgctcc cagagctgac cttgggttgt gagctcagct 41640 ggggcgtgcc tggggctcct ggattgccct atttctgtca ttaccctagg tattctagag 41700 ttaaggggag cctgggttga ggggatggtt ctggctgcct ggggaggtag gggatgtgaa 41760 cactgtggta tcagcattga gccctggaga cccttggcct tgatcagcac ccccagagag 41820 tccacaagag agcttctgag gctggtcatg cctaataacc atcttcttat gaagagcatt 41880 aagacatttc taggagcttt tcaccagaaa tggctcttta tcttcccttg tgaggagtag 41940 caatgcaatt atagccccaa tttacagaca gacctgcagg ggaggtgtag agcaggaggt 42000 gacttgcctg ggattcccca gtgggttaga gatgaagccg ggatggagct gaggaggatc 42060 ttgtggtgac aggagaaggg tccagccatg actgacctta ccactcacag cctcccggaa 42120 gcaaatcttc aagagggccc aagtgggagc cctgggtgat tccaagagaa ggagccagtt 42180 gtgcatgaat ttggagattt ttctcaagga gtcattttct tgggtctcct gagcactgag 42240 gaaaggagga agacatcatg gggttggtgg taggggcagg cggcgacaag gaaaattgtc 42300 cttgggtgaa ggactaaaga taagaataaa aaacaaagtc tttttgttgt tgttgttgtt 42360 gttgttgttt ttgagatgga gtctcgctct gtcacccagg ctggagtgca gtggtgcaat 42420 ctcagctcat tgcaaccttc cacctgccgg gttcaagcaa ttctcctgcc tcagcctccc 42480 aagtagctgg gactataggt gcgttccacc atgcccagct aattttttgt atttttagta 42540 gagatggggt ttcaccatgg tggccaggct ggtctcgaac tcctgacctc gtaattcgcc 42600 tgccttggcc tcccaaagtg ctgggattac aggcttgagc caccacgccc agcctaatca 42660 cctgtgtctt aacaagccct ccaggtgatt ctgacacaca ctgaaaatta aggaccactg 42720 ttacagaaga aaaggatgtg gtatcggtat gctagggctg ccttaacaaa acatcaaaaa 42780 ctgggcggct taaaacaact gaaatgcatt cactcatagt tctgaggcca gaagttctaa 42840 accaaggtgt cagcagagct gcgctacctc tgagggtgct ggggtgggtg cttctttgcc 42900 tcttccagct tccggcagct gctggcgttc cttgtggctg catcactcca gtctctgcct 42960 tctgccttca cgtagctttc tcctctttct ctctgtgtct ctctgtggtc tctgataagg 43020 atacttatca ttggatttag ggcccacctg gatcatcctg accatctcac cccaagatcc 43080 ttaatacatt tgcaaaggcc ccttttctaa ataatgtccc atttacaggt tctggaagtt 43140 gtatatcttc tggggcctcc atttcaccca ctacaaggcg aggagggaaa gtgggcctgg 43200 accctgtgag agagcacttg ggggtaagaa gctgaggcta gagtcaggaa gaaaaacagg 43260 agggttagag tgggcagtag tcacgagccg gctgtgccca ccatgggggc atctggccaa 43320 gtgaggccca cagctggttg gagaagggag caacaggggc aacaaggcca gaggtctggg 43380 gtgctctcat ctaaggcctt gtcctgaagg tgccgtctgc tgtggaaaag cctccccgcc 43440 aaaatctgcc tgcaagctgt gcaggtgatc tagtgaaccc tgttggcacc acccatcctg 43500 cctgccagcc agagggccct aaggcccagc tgtgctgtag ggcccagccg aggggtcccc 43560 agggacgcac gaggtggcag atggcctccc tggcatggct attttgggct ccgtgattat 43620 atgctactgt atttggatct tcccaaggtt ccgggagttg attaccacat acgtttgggg 43680 cagaggctgc tctgaggtgg gtgggtgggc gggtaggcag ccccctgcag ccctcaaggg 43740 tctgaacatg ggtaatttgg ctcctgagag atgctaggag agtgttcgtg gtgggctggg 43800 ccaggggatc aggggtgatt ctttcagccg taaatgccgg tctgggacag agttggccca 43860 agtcgggcca cccaggagca gtgcgggaac acctcagctc agacttgtgg ggctcctcag 43920 ccttcctgct agccctctct ggctatggtg ctagggcttt cttgggccag aatgttctcc 43980 tctgactggg ccactcctgc cctctgcgcc ccacagagct caagcaggcc aaggcctgag 44040 aggcctgagg caagagtacc ccgggtgttg cgaactgata gctcagattt ggtgtaggtg 44100 caattaagcc cccagtcggg accaggaggt gggtgatccc tcccccatgg ggagaggaca 44160 gaggcaggct ttctcctcag ctctccttcc tcttgtatta ttgccccttc ctgagcagct 44220 gatgccagtg cagagccctg gcctgagttc tgggtctgtc aggagggaag aggagaccga 44280 actattggtg tgtctctgtc catccatcca ttcatccttg catccatcca tgcatccatc 44340 catccatcca tccattcatc catccctccc tcccttcatc tatccattca tccatccccc 44400 atccctcctt ccctctgtcc ctccctccct ccctccatcc atccattcat ccatccctcc 44460 ctcccttcat ctatccattc atccatcccc catccctccc tccctccctc tgtccgtctt 44520 tccatcaata tatccattct ccatccccat ccctccttcc cccatccctc catccctcca 44580 cccatccatc catccatcta ttcatacatc cccaatccct ccctccctct ctctttcaat 44640 ccccaatccc tacatacatc catcccttcc ttcctacctt ccccaatccc tccatccctc 44700 cacccatcaa tcaatccatc cattcatcca tcccccatcc ctccctccct ctctccttcc 44760 atcccccctc cctccatcca tccctccctc cctcccttca tctatccatt catccatccc 44820 ctgtccctcc atccatccct ccatgcatcc atccccccat ctgtctgtcc atctgtttgt 44880 ttttgtaaac tttgacgagt gcttgaaatg cctaacacaa gagatactga gataaataat 44940 aaaacaaggg ccctgccttt tcagcagctc ttggtctgat gtaggaatat tagatacacc 45000 aagaccccat cttcatcctt cccttcccat acattctccc attctgtgca cttaaaattc 45060 tgtgacagaa aagtcagtgc aagcaaacat gatgtaaaat atgtatataa aagctaaaag 45120 agaattgact aaaatgatga aaaccgggtg aagcccagtg tgtgtaataa aaatcatatt 45180 ggtagttaat gtttggtgag tgcttactat gttaccaggc tctaggctaa tccttggcag 45240 actacctcat tccttgcaac aagcctctaa aggaggtcct gtgatccttt ttttacagat 45300 gaggaagtga ggatgtaggg aattgatcag ttggtaaaat ggttcatctg catacaaagt 45360 tcatgcatgc tgttagccat gactttagac tgtgcagaag ggcagctgtg agggaccctt 45420 gaggtctagt tcccaaggct gtctacctca cagcactagg gaaactataa agtgcagatc 45480 cccaggcccc ttctcagact tagtgaactg gaatcttcag ggctggggcc caagaatctg 45540 gatttttaaa aaatcttccc aggtgattct gatgcacagc taggtttggg agccactgct 45600 ccaatcctcg ccactcaaag tgagcttcat tggtcagcag catcggcgtc acttgggagt 45660 ataagtgcag acctgctcac gcagattctg cattttaaca ggacccatcc ctccaccccc 45720 caggggatta gtatgcaatt caaaattggg aatcactgtt cttttgttca ggagcttctg 45780 ggacattctg cgccctctcc caacctcagc tgagatggac agcaatcatt atcagctgtc 45840 cataggtctg tcccctaggc cctaaggact ttctgcagaa agtctccaac ccttagaacc 45900 gcttttttgt acccccgcag tcacttgtac acatgggatg taaatgttaa ggtttagact 45960 ggtttggttg tggacccact tggaggcagt gaaaggtact agataacctt ttgggcgatg 46020 cagttgggca gataaaggtt agactttggg aagaacatcc tagctgtcta taccacacaa 46080 aggggaaaag gctagagtat cttcttggga aggtacatta gagtcacact ctcctcacct 46140 ggcctggggg caggagaaag tgtatgacga tctcactctg atcccatcat gacgttttct 46200 gggagtatcc tcagagaatc ccagagaggt aagagaaggc tcaggctgca gccaggaaga 46260 gtggagccag cacctgtggc ctgcagcagc cttccactgg ccacacccct ggagcctttg 46320 cctgccccct cctcctcccc agcgcctcag ccatcctgat gagagaagaa gggtcggggg 46380 ctgggaaagt gtgatctttt tcgagagcgt gtgagtaatt ttccatctgt ttatgagctg 46440 ttggagccca ggtgtctgcc agccactcgc tggagggtga aaatccacac agcaaccgga 46500 aaaggcggca agcacaccag gctccttccc tggccaccgg ctgaggctgc gtgggtggag 46560 gctgccctcc taagaccaca ggaccttaga atactggggc tggaaggcgt catggaggtc 46620 actaagtcca actgactctt ttacaggtga ggaaaccaag cagttgaaag aggaagttac 46680 ttcccagtgt cgcccagtga atcagtggca gcgctgcagc tagctggaac ccagaccagg 46740 cctgcagatg cttttttggc tctggaaggg gatgatctat ttatgagcct ctgtgaccac 46800 tgcaaccaga tggctctaag agcagttgtt ctggaatgtg accccatccg ggggactctc 46860 gccaaagtcg cttccctccc ctcaccaccc tgacacctgt tttgagactt ggagccagct 46920 ccttcctttg ccctccctta aacgactcta gcaaggcctg gtctggagga ctttgagggt 46980 cctgggaggg gtctggacta tggagaggga ggagcgaggc ccatggtgtt tggggggatg 47040 tgctgctgtg gcgtggcagt cactgaaagg ttgcagtgcc ttcagggccc tggcagccca 47100 cagagaggac atcattgctg tcccctcgca aagcaatctc ttccccaagg aaaaaggaca 47160 agtccagctc tctctgccct ccccatgttt cctagaagaa ttccactttt tctccagaag 47220 gtgtcctgcc tttattgcct ggtgagtggg gagtgcagca cctttctggg gctggaggcc 47280 acagaggacc atcagcacag gtgaccctgg ccccctccct atcctgggag ccgaggcacc 47340 acagttggga aagaggagag aaggcaggag ttggaatgaa gggaaagagg aaagaaactt 47400 tctttgagca tctcctgcgt gatagatacc aggtaccttc ctcaggctga tctccattca 47460 gcagcagggc caccagtacc actcaccgag gctccactgc agccgactcc tgacttcgct 47520 ctctcctccc cttcacagtc cacaccagtg gtgtccatca gttcttcatc tacaggcgaa 47580 gcattcttac ccttctgata aggtgctttc cagcccttat cttatttgat tgcataggag 47640 catgtgataa tgttgcctcc tctttcctta aagtcctttc ttcctgtgcc ttctgtaagg 47700 agaccccatt ctcctagatc tctcctctct agtcactcct cgattctcct tggaagctcc 47760 tcttcccctg cccatcctat aaatggcaga attctttggg agctctgccc tatctctgct 47820 tcagtttctc cctgggaggt ttctcatctg cccatggact cagtaactac ctgtgtactg 47880 atggttctca agtcttagct ttagcttaga ttggtacaat gaacggccta ttggaatctc 47940 cacttgcatg actcacgggg cttcaaactg aacatttctc aaagtaaacc catcatctcc 48000 tatgcatatt ccttctccac atcctctgtt tcgagaaata acatcattta gttccctaat 48060 tcggagactt gggtgtcatc cttaactctt tcaccactgc atccaatcaa tcaagtccca 48120 gtgactgtac atcctgttcg ctctggagta ggtcctcttt tccttacttc ctggtggcca 48180 ccattgtctg cccgccatca tcacttgcct gggctgctgt aacagcctcc ttacaggccc 48240 ccagcttcta ctctggactt accccagtct tttccccaca ttgcagccag atggatctta 48300 agacacgaat gtgatcatgt cactcccctg cttaaaattc tcagttggct cccaattatc 48360 ctgaagacaa agcccaaact ccttggctaa tgtacaaggc aacccacaac ctggctcctg 48420 cccatctctc accacttccc ctcccactct atgctgtagc cagactgagt aacttgcagt 48480 tctcttgggc gtgcagcctg tgctcctgtg ctttgctgtt ccctctgcct gatatacttg 48540 ctcctagttc tccttctcac catcctaatt catccttccc acctcctact catcctttag 48600 gtcttacctt gaatgtctcc tcctgaagga ggccttttct gattcattca gctgcctctg 48660 tgctgcactc ccagaacagc ctgcaatcct actgttattc tcttatttca tcctgtttcc 48720 ctctatactc tgagttctac agggttggag actgagtgtc cttttttatt ttttaattta 48780 ttttttattt ttattattta ttattattat tattattttt tgagacagag tctcactctg 48840 tcaccgaggc tggagtgcag tggtgcagtc ttggctcacc gcaacctctg ctttcaaggt 48900 tcaagcgatt ctcctgcctc agcctcctga gtagctaaga ttacaggggc ccaccaccac 48960 acccagctaa tttttgtatt tttagtagag atggggtttc atcatgttgg tcaggctggt 49020 ctcaaactcc tgactcaggt gatctgccca cctcgcctcc tacagtgctg ggattacaga 49080 agtgagccac cacacccaga ctggatgtcc ttttttaaaa ttcaggattc cccattgcta 49140 gtacaatatg tggcacatag taggcactga gtgatctttg cttataattg tacattttaa 49200 tgaatgggga aagaggacca gagaggttaa gtaatttgct cagtaacaca caatctgtaa 49260 gatctggagc ttgaatttaa actcaagtat gtctgattcc tgagtccatg ctttttctgc 49320 cataactccc tggttccccc ttcctctaca ttcttcctca gaacacccaa gaagttctga 49380 ggaaggatgg acctggggga gaagggccct gcccgctctc tgctgtcgtg tttcggggag 49440 gtggcatagc agacagagct gagaggggga gtaaaagcag gaaagctacc caaggggttt 49500 aaaacgagac gatcacagat gacggcctga tataggcaat gctgttggct tccagttgca 49560 agcactggct ctctgtgctg tgaattcaca gagagaaggt gtatgaggag gctgcaggcc 49620 tccactgtct gatagtgtgt tgtcagagcc tgcacgctcg tatgtggatc accctaaata 49680 aaacaggaat gagaaaagaa actcgttatt ttaaaaaagc gatacagtgt cttctgcagc 49740 caacaataac ataatttaga attacctgct atatgaggat gatctatgtc cctgcagccc 49800 ctgagccttc atagggaatg gagaggcagg agctacaggt acagcattga agactacata 49860 ggggcattca ccatttccag agctccaggt tggggtatgg ggggcaggag agggaagaag 49920 aggaagcctc agatccctcc cacccgggaa gccaggaggc tctattcccc aggcaggact 49980 cccccagcca tttccccttt ccttcctgct gtcagctgca gctgacagga atgaggctgc 50040 tgggctcctc ggggagccac caggcctttg gggccaatgc agctgcctct gcccccagcc 50100 ccaggccagc atcctcccct ggcccccact agcctgcctc tcagctgcac atctgctaac 50160 tgggggcctg aagggcctta gagtaaaacc cccctcagaa catctggagt gtggggggct 50220 ggaggggagc ccacctggaa gggagaggag atgggaaagt ggagagggaa aggggaaaga 50280 gagaagagga gggagaagga ggactgttga gggaggggcc agactgaggg gatgaggggg 50340 agattggagg caaacaaata gtccccggat tcttagttct gccttttgtt gaggtgaaat 50400 gggttggagg gggtgtggtg aagggggtgc ccagggctcc tccaagtcat caaatgacac 50460 cacttacgtc atctcactcc tctgatcaga aaccgcccac agaccactga ccagctgggg 50520 caaatcttcc cctaaattat acctgttttc acctttgtgt ccctataccc tgctctctac 50580 tccagcgaag ccacttccct ttcttccccc tacctcccca aacttgtcct tctacctcca 50640 tgcattggct taagctgttt ctcagactag gaagtctttc cttttccttt ctgccaagcc 50700 ttgactatac tgcctgcatc aatacagccc tctcctcctc caggaagccc ccctagaata 50760 attccatctg actctgggcc tgctggggaa cactaaatac cagagccagg catcctcact 50820 ctttataatt atttcttgtc tcttagcctt ggctttccca tctgtaaagt gggtgcagcc 50880 cctgggctcc tgtacgtgaa aggtaccatg ggtgattagt ggcaggattg ccaccagctg 50940 agccaaatgc cctgctggac cagagtctgc cagcaactac ctgctgccag gggcatggag 51000 aagaacctga caagaattca ggttgtggaa aggaacaggg cactgacaat acatagcctt 51060 taggtactgc ccagaaatct ggtttcggac ttgtgggatt gggacactaa atggtagcct 51120 tttggcaggt gtgaaaagtt tggaaagggc tccatgccca gcccatggca gatgggtatg 51180 cttcctgtct cctttctctc cctgacaaga gtcctgaatc tgactgtgtc cagactgctt 51240 tgagacttga aagtcagagg gaagaaggaa agtaagcctt agggagcatt tgctaggtgc 51300 caagcattgt gtcaggcaca tgcatgggca ctctcaccac actccttaat tgagggcttc 51360 aacgcaccca gcaggaagac attctgacac tctcgagtca tggctcagtc tggggactcc 51420 agactgtggc agcccaaata atctgtctac ttttagctgg aagggtgatg tttgatttta 51480 gggtctctga aaatagtgct gcttttctgt gaaaaccagc tctgccattt ctagataaag 51540 tatcataacc aaattgtctg aacattctga gcctcagttt tgtgatctat aaactaggaa 51600 taacaaagtg gttgttttga agactcatga gatgacgtat aggaagactt agcacaatgc 51660 caggtacatc ctgagtgcat gataactgat ggtcctaata ctcttttttt ttttttaaga 51720 tggagtcttg ctctattgcc taggctggag ggcagtggcg cgatctcggc tcactgcaat 51780 ctccgcctcc tgggttcaag ctactctcct gcctcagcct cccaagtagc tgggatcaca 51840 ggtgcccacc accatgcctg gctaattttt tgtattttta gtagaagcgg gggtttcacc 51900 atgttggtca ggctggtctc gaactcatga cctcgtgatc cgcctgcctc ggcctcccaa 51960 agtgctggga ctacaggcgt gagccaccgc gcctggccaa gatggtccta ctattctttt 52020 tttttttttt tttttttttt ttgagacgaa gtctcactct gtcgcccagg ctggagtgca 52080 gtggcccaat ctcagctcac tgcaacctcc gcctcctggn nnnnnnnnnn nnnnnnnnnn 52140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52740 nntgttctta ttagactaat ctccagaact ggctcctacc acatcatcat tttccaccag 52800 ggtgggagcc tgaggcagga tgctttgtgc agctcaggga aactctggct gtgaggagct 52860 ggagtaggat agactggagg ggttcctcac ctctggtcag gctttgctgc aaaactagat 52920 tatccaggaa gggaaagata cttcctccac cgctgacctc aggaatggtc cgtctggtgg 52980 tgagttgccc ctgccatgag tgaatgtggg tcattccttc cattatgttt gcattctgca 53040 gattctgtgt tgtacacttc cccatgccag gtgctgatgt gcctgccttc agtgagtttg 53100 ttaccaaata agggctgtga gacatgttca taaatagtac atgctagaat aaatatagct 53160 ggtgcatatg caaagtccag aatgctgtgg gaaggggagg agcaagtgtt tgctgggtga 53220 gtgtgccagc actgtgccag ttttgaaggt agaataggag ttgaactggt ggacgctgaa 53280 tgaggtggct catgcctgtg atagtatttt gggaggctga ggcgggagga tctcttgaac 53340 tcaggagttg aagatcagct ggggctgtaa caatagttga gactctgtct ctacaaaaaa 53400 aaaaaaaaaa ggaaagagaa aagagttgaa aagacagttt ttgctaggag ggaaaagcct 53460 gagtgaagtg tgaaggcagc gaattggggg cgcggggatg gggtgtgaca ttgctttaga 53520 gaatggcagc ttggtttggt gcagacacag cttcctgtgc ttgtagcact gtctgcctgc 53580 ccacttgaag gaagacctgt gtgtaggaga taaatctgca gagagggccg tgacttttga 53640 tgaccagcta aggactgcct cacaggcaag ggaggttcac cacagggttt tcgtggagac 53700 agatgtgtgt cccagaaaag cacttggacc tagctgtcca tagtggaccc cagagcttct 53760 gccatgggcc acaagtgagg ccagttagga gatcacttca ggaggggtcc tggaccacag 53820 cagtggcctt ggggtcagga ggagcgggca tggccttgag attccattac caggacctgg 53880 tgactgattg tataggaaat ccaggcaaag gtgggacaca gagcaggtga aatgaagcaa 53940 ataaatccct ttccactgcc tgctcctcgt ctctccctcc ccagcgccct tccccgctca 54000 ggctgcagag tccgtggagc actcccttgc cggcccccct tagctcagct ctccaacagc 54060 ctgggagtta attatacact gacgtacttt gtttttaaaa acatgtttat ttttgctctg 54120 actcattgta gaaaatttag aaaatacaaa aaataaaaag aagaaaataa aaatcaccca 54180 caattctacc acccagacac aatgcttgtt aacatttctg tgtatttctc tatgagcaca 54240 catactctta caaacacaca tgcttccttt gtgatacatt aaatctttac atattatgtc 54300 agcatctggt ttattgtctc tttaaaattg tgcgagtaca tcactagtgt aagactttta 54360 gctttaaata tttaacaatt gagatataat ttacacagca taaagttctc tcttttaaaa 54420 tatgcaattc ggtgattatt agcatattta ccaagttgtg caactatcac caataagtct 54480 agaacatttt ttgtcaccca agaaagaaaa tccttactca ttagcagtca ctctctatct 54540 catcttcccc cagcccctga caaccagtaa tctgctttct gtctctttgg atttgcctgt 54600 tctggacatt ttatataaat ggaataatac aatatatagc ttttaatatc tggctttttt 54660 tacttagcat aatgttttca aggttcatcc atgtgtatat ttatgcatgt aacagcactt 54720 tattcctttt ctattttttt taattttttt atttttgaga tagagtctcg ctcagccacc 54780 caggctggag tgcagtggtg cgatcttggc tcactgcaac cactgtctcc cgggttcaag 54840 cgattctccc gcctcagcct cccaagtagc tgggattaca ggtgcatgcc accatgcctg 54900 gctaattttt gtattttagt agagacgggg tttcaccatg ttggccaggc tggtcttgaa 54960 ctcgtgacct caggtgatct gcctgcctcg gcctcccaaa gtactaggat tacaggcgtg 55020 agccaccgcg cctgaccacc ttattccttt ttatagctga attatattcc ttggtataga 55080 tataccacat tttattttac ctctgtatca gttgatggat acttgggctg tttccacgtt 55140 ttggttatta tgaataatgc tgctaaaaac atttgtgtgt aagtttttat gtacacatat 55200 gttgtcagtt atcctgggta tctaactaga aatggaattg ctgggtcata tagaaactct 55260 atgtaaaact ttttgaaaca ctgtcaaact cttttccaaa gtgactgcac cattttatat 55320 ttctgccagt attgtatggg catttcaatt tctccacatc cttgccaaca cttgttatgt 55380 ctttttgatt atagccatcc tagtgggtgt gaagtggtat ctcgttgtgg ttctgatttg 55440 catttccctg atgattaatg atgttaagga tctttttgtg tacttattgg ccatttgtat 55500 atcttgtttg gagaaatgtc tatttagatc ccttgcctat tttctgactg ggttgtcttt 55560 tcattgttga gttgtcacaa ttctttatat attaaagaca taagtccctt atcagatata 55620 tggtttgaaa atgtttttct cccattctgt cttttgtctt tttcactttc tttatcatat 55680 cctttgacat aagtccctta tcagatatat ggtttgaaaa tgtttttctc ccattctgtc 55740 ttttgtcttt ttcactttct ttatcatatc ctttgaagca caaaagtttt taatttcaat 55800 gaagtccaat tagtctattt tttggtttgt ttgttgtact tttggtgtta tatctaaaaa 55860 accattacct aatccgaggt cagaaagata tatgcctatg ttgtcttata aggatttaac 55920 agttttaggt ctttaaacca ttttaatttt tgaatatggt tcatacatac acaatgtagc 55980 tagaggtcca gctacattct tttgcatgta gatatccaca tgttccagca caacattttt 56040 gtcatcccaa atgctattct ttaaaaatgc tgttttctcc tattgaattg tcttagcact 56100 gttcttgatc atcaattgcc cataaatgta agaatttatt tctggactct gaattctatt 56160 caattgatct acatatctat ccttaccata tagggctata tggtctgcat tactgtagct 56220 ttgtaataag ttttgaaatt gggaagggtg agtcttccta ctttgttatt ttcaaggttt 56280 gactattctt gctcccttgc atttccatat aaattttaga atcagcttgt caacttctga 56340 aaaaagacca ctaggactgt gatagtggta gcattgaatc tgtagatcaa tttgttgagt 56400 attgccatct taacaatatt gtcttccaat ccatgaatat gggatatttt ccgatttatt 56460 taggtgtttt taaatttctt tcaacaatgt tttgtcattt tgagagtata ggttttgcac 56520 tgtttttgtt aaatttattc ctaagtattt tgttcttatt gatgctattt taagtggaat 56580 tgttttctta atttcttttt ttggaatact cattgcaagt gtacagaaat ccaattgatt 56640 tatatatatt gatcttatat cttgcagctt tgctgtactc atttactaga tataataatt 56700 tctttgcgga tttttattaa gtttctaatg tttaatagga gcagtccctg ttcatttttc 56760 tcttatattt tgctcatcta tttattatat gacatgaact ttagttttat tttatgaagg 56820 tgccgtcact tcctgccaaa aatgtcccac tgggatctta attggaatta cattgtctat 56880 aagttgattt gtagaggctt tacataatta aaaaatatag tcttctccta caagatttat 56940 tatttctctt aattcaaaac atcttttatg tatctcagta atatttttat agtttttgtt 57000 ttttttcatt aatgactcat ttcttgttag aattactcca tgatatttat tgggttgtgt 57060 ttgtttatga gctttctttg gtggctattg ctaatgtaaa gggggtattt tcatcattgt 57120 ctcttttatt tattgttgga atttaggaaa atattgaggt gtatacattc atcttgtatt 57180 tgatcacttt attaaattct cttattaatc ttaaggcttt atcattgatt ctcttggaat 57240 ttatagatat ctaatattat ttgtaagtaa taattatacc tcttatttct gtttcctgtc 57300 ttatgcgtta gagctttcag aacaatgact attagcaatt atgacaatga acatatttat 57360 cttgttctgg attttaagag gaagatctct agtattttct cattaagtat gatactggat 57420 atcctttgag ataatagact ttaccatgtt gagaaagtat ttctctgtgt caatatttta 57480 cattttttta aattagaagt tttaaatgtt gctgagtggt ttttttagta tatattatgg 57540 ttatatatac taaacccatt atactataat agaatttatt attttaacat cacagtattc 57600 tttggataat cccatcttga tggtggtaga ctattcttac tgtgttttat tgccagtatt 57660 gtaattacaa tatgtacatc ctaataaaat attcataaat gagaatgggc tatagcttgt 57720 tttctgtgct ctctttgatc acttttgtta ttggtttaaa ctgagttcca agaatgaatt 57780 gagtatcatt ccagtttttt ctatccctgg aacaatttat agagcatgaa aattattttt 57840 tacttgaaag tttggaaaaa tttactgaca aatttcctgg ccccagaacc tgtgtttgaa 57900 gagcaaattc tttgataaca tttctaattt cttctttagt gtttgatctc tttggttttc 57960 tacttcttag atagttttga gcattatctt ttcctgaaaa ctatccattt catcaaaatc 58020 ttttccatct gtgctaatat attaaattat tttaagaaaa tgagttgtat cctggaaaga 58080 tgtaaatgat gctttgtctc acgtctatca tgttttatac tttttaaagg cttaattgat 58140 gtttccattg ttctccttct tgccttctcc tcctccttcc tcccatctct ctttccccct 58200 ttccctccct ctctctttta ggtactctat ccctcaaatt ttagccaccc tggcttccct 58260 gaactctgca cgctatctcc tcaacttaga gtgaacacaa gttctgcctg agttccctct 58320 ctttgtcctg cagcctggac accctctcca ggcaaaagcc tggccaatca taaggctcac 58380 agaatgtgcc tcccggctgt cagcaatcac ggtcctgcgc tgcctgttgt ccaatgcctc 58440 aaacctgttg cttttcactt gtttgaggca aaaacaactg ctactccaat cctgcctgga 58500 agtaaaagcc aagaagaatt ccccattatc tcattttatt ttattattat tatggttttc 58560 tgagacggag tctcactctg tcgtcccggc tggagtgtag tgtcttgatt tcggcctcag 58620 cctcctgggt tcaagtgatt ctcctgcctc agcctcccga gtagctggaa ttacaggcat 58680 gtgtcaccac gcccagttaa tttttgtatt tttagtagag atggggtttt gtcatgttgg 58740 ccagactgat ttcgaactcc taacctcagg tgatccgccc gtctcagcct accaaagtgt 58800 taggattaca ggtgtgagcc accgctcctg gactatttta ttttttcaga cagagtctcg 58860 ctctgtcgcc caggctgtag tgcagtggca caatcttggc tccctgcagc ctctgcctcc 58920 cgggctcaag tgattttcct gcctcagcct cccaagtagc taggactaca ggcatgtgtc 58980 tccatgccca gctaattttt gtatttttag tagagaaagg gtttcaccat gttggccagg 59040 ctggtctcga actcctgaca tccgcctgtc ttggcttccc aaagtgtcgg gaatgagcca 59100 ccgtgctctg ccaaattccc cattcttgag ttcttcattt aaaaattatc tctcttgatt 59160 gcctgtagta tattttcaag tagtttttcc cccaagaaca acagatggca actgtatttc 59220 tgatctttta tatagttgag aatatatttt tttgtggcca actaactaat atattttttt 59280 gtggccaata taaaattatt ggatcacaat ctatttttct cagaatcctg tagataaggc 59340 tccatttccc cctgacatgc agtgtgccag gaaattctga ggtcagcctt atcattttat 59400 tctttcaagg agagcatatt ttttctttat gaatgcttac aagatttttt ttctttatcc 59460 ttacaactga aatgtttgcc agaatgtgtt gaggtattct ttccacttag tttgcttggg 59520 atacaataaa catcttaaat ctacactatt ctttcagctc taatgttttc tttttattat 59580 tattattatt attatacttt aagttttagg gtacatgtgc acaatgtgca gcttagttac 59640 atatgtatac atgtgccatg ctggtgtgct acacccatta acttgtcatt tagcattagg 59700 tatatctcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 59880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnng ttgcgaaatt ttctcccatt 59940 atgtaggttg cctgttcact ctgtaggtag tttcttttgc tgtgcagaag ctctttagtt 60000 taattagatc ccgtttgtca attttggctt ttgttgccat tgcttttggt gttttagaca 60060 tgaagtcctt gcccatgcct atgtcctgaa tggtaatgcc taggttttct tctagggttt 60120 ttatggtttt aggtctaaca tttaagtctt taatccatct tgaattaatt tttgtataag 60180 gtgtaaggaa gggatccagt ttcagctttc tacatatggc tagcccatat gttttcccag 60240 catcatttat taaatagaga atcctttccc cattgcttgt ttttctcagg tttgtcaaag 60300 atcagatagt tgtagatatg tggcattatt tctgagggct ctgttctgtt ccattgatct 60360 atatctctgt tttggtacca gtaccatgct gttttggtta ctgtagcctt gtagtatagt 60420 ttgaagtcag gtagcatgat gcctccagct ttgttctttt ggcttaggat tgacttggca 60480 atgcgggctc ttttttggtt ccgtatgtaa tgttttcttt cagttacagc tatcatgtgc 60540 ttctgctcca ttcactctgg gtttaatcct ctggaatacc tatgattctc aaattcaaat 60600 tcagttctcg tttctatcta tctcatattt atttccataa tcttcgcccc tttgtccttt 60660 tattctgact tcaaggagag tgtctcatgt ttattctccc cattgctaat tcagtttttt 60720 gctgggccag gtctactcat tatcacctac aatgcaaatt tgaattttgc ttttttaatt 60780 tacttcccct gcagtcttcc catatctcac ctagttccat ttacacttca atttgtcttt 60840 gaaacttgcc ttttatttta ttttatttta ttttatttta ttttatttta ttttatgctg 60900 ttgttcttct gtcttagctt tttttctttt catggatttt tgttcctgtt tcagagaggt 60960 catgatttct tgtaccttac tgagggtgtc aaataacttc tactacaccc tcaataaatt 61020 attttcagag ttatgatcat ctttttttct tttctgagac agggtctcac tctgttgccc 61080 aggctggagt gcagttgtac gatcttggcg cactgcaacc tccgcctccc gggttcaagc 61140 aattctcctg cctcagcctg ctgagtagct gggactacag gctcataccc caatgcccag 61200 ctaaattttg tatttttagt agagacgggg gtttcaccat gttggccagg ctggtctcga 61260 actcctgacc tcaggtgatc ctcctgcctc agcctcccaa aatgctggga ttacccacat 61320 gagccaccgc gcccggccag catacatctt tttgtttgtt tgttttttct ctatatatct 61380 tcataggtgt tttagtgaga aggtagctaa gttttcctcc attttaactg gaaacaactt 61440 gagtgtgttt gatttgttcc ttcatttcct ccctaggcta gaggagaggg attatggctt 61500 cagcacgggg gtgtgggtgt ggtgggtact tgtggaccaa tcagtaagga cttggaagat 61560 gctgagtata ggagggaaca gcaccatgtg aggttaggtc agaaggctct agaaagagga 61620 gttttaagca gagctttgaa gagaggatgg agaaacatct ttcaaagagg ggcatcaacc 61680 tatacaaaga tccaaagatg aataaactat gtactgtggg acaaagaggt aaggcaagag 61740 gcacatttgc atatgaaata gagaactggg ggagatcttc tgagaaatta tgctggtaag 61800 acagtcattt cagggaaggg atcttgaagc ccaagccaaa gagtttgggg ttgactagaa 61860 gccatagata attctgagca agagaaaaac aagattccaa ccatggggat gattgtgtag 61920 tctgtgtgag tggatggacg gatggatgaa tggagaaatt gatggatgga gggatgtggg 61980 gccaggaaat agggaggctg gggaggggag ggttgaaact atacaggtat aacctctcca 62040 gttctcatac tggtcacaga gagcctggga gctcttggga atgagggaga aaatagattg 62100 tgggaaggac tggaaccctg tgggaagcca tggttgatat ggacttttcc catttcttct 62160 ctcccttctc ctatggtact tttagaagtg ttaatatgtg cctgaatttt ccctggtgcc 62220 tattactaca aagtctgcta ggacagcttt taagtatctt taaaactctg ggatatcact 62280 tctattccct aagcctctga catcactgcc ccctttctgg agaagcaagt cgggaagatc 62340 tccatcggcc tcttcctcta ccaaccacag ccaccctttg ccagggcagg cagccaggcc 62400 tcaagcaacc acaggacccg gctcccaagc acatctcaga cagcccacga aggtcgccag 62460 aaaccagcct gatgtctgca attcaatggc tggaggctgg gggatggatg agatggcttt 62520 ttgtagttcc caccccagga ttccgggatt gtcacccaca aaggtggcct tctgcccaga 62580 tctgtaggtc tgtgtctttg ggggaagctc ctaaaggcta cacacagagg cacttccctg 62640 tctccctcct ggagggtggg taattatttt tatatattgc tgctgaataa aatacaagga 62700 atagcttgga ggaatgctca ggtatcatcc tttgatgtat acaaaggaag cactttctct 62760 gcccgccccc gccaccaccc caggggttct tgcacaccca tcaatgctcc tgacagatgg 62820 aaggaaagga gagttgaggc attagcatag ccaggagaac cagttggaat tgccttcgtt 62880 ttaatatccc acctttgatc tattgagagg ctgacagtga ggggaaggat cttattagat 62940 ggcataaatc aaagctgatg aaatcacttt cctccaacaa gcttttcccc tgggtgagga 63000 gagagagaaa agtgctgctc tagctcccac ctcccctctc tcctcaattg taagtgggta 63060 tggaaaggtt aattcaaggc tccatgcacc atcaccccca ccccacctct gcagggaagt 63120 ggagttctca ctccagcttc tggtggctca gttgacctcc tctgctcccc atgctctctg 63180 tcaccatggg actgtcccac caggagtact caggctccga ctccccagag ttgtcacttt 63240 tgggctcttc agggcttgtg tgctctgtga gggcctggtg atgggttcca gatggggaga 63300 ggcactgctg ggatgcaagg gatgagcccc aagtgttgga tgctgccaag gaatggttct 63360 cagagaccag ggtcttcata gttcctcaac cagtgacctg ggaagtgtct ctgggggccc 63420 cccattgcac tgtatgtggg ggtgggcagg aatcggtgaa caccttcttc ttctcccaag 63480 aggacaaggg caggagagag gtgaacagtg aactcaaaga acaccggact caaaaccaat 63540 gccttctctg gctttgccat ctctttactc tgtgaccttg gacaaatcac tcagtccctc 63600 agagtcccct ttcctccctg gttaagtggg gtccctgtta gctgtcactt ccactgctgt 63660 gatctcccca tcaagcaatc atctgaacaa tgctgaaagt gcccataact ggatcaataa 63720 aaaacacaga ctgaatatcc atggtgggca agtttcaggg aaagagggga catagcccgc 63780 gtcttcaggg gtcttcccat gtaatggggg agaaaagtga ccaaagtggg cacttgggta 63840 aagttgttca aagctctgaa gttggccttt tactatctcc cacctctatc gaccatcttt 63900 cccagctcct catctctctg gtccaaagct ccctgtgttc caagtctgtg tctcattttt 63960 agaagcttct ctcaaaaagc tgttctccac taaaaggcta cctaataata ctttccagat 64020 taaaaaaaaa aaaaaaataa caggcagctt ctacactcta aaaaaagaat attatccaaa 64080 tggtagaatt tggtcatctt gagatgcctg gaggtggggg agtgggggtt ggctactctg 64140 tgcctctgca caacaccatg tgcttgtaag ttgccctgcc tggggacaca gggacatagc 64200 acagaagaat ttgtccaaag aatgtatagg ctctcggacg gacagagcca cagggactct 64260 tgaagcgaac ccctccttga gcaggtgaag acactgatcc tggagagagg aaagggactc 64320 gtctgaagtc atagcgccct gtgtatcagc aagaacaaga cctttgccag ccttcacatc 64380 cccaccccca ccccaccaga gtggtctttc tcaacccaga cttcccaatg cctacaattt 64440 aaagttcaga tccataaagc ccccaaggac ccgcacgttc ttttccctac tacctttcgc 64500 atctgaccca agccctcact ccctgcgtca cccaggaccc tctgctcagc acagatgagt 64560 tgttctcctc tcccatgttg ttctttggtc agtacctctg cacacaccct tctttgccag 64620 aaatgctcat taccctcatc cacttggtga aaattcagtt attcctcaag actcacatga 64680 atgtcccctc tttactcctt ctaggaagtt tgtcctacct ccgtggctga aggttccaca 64740 ccctttaatt cattccctca actcttggtg cacaccattt tcagacacag cctctctttt 64800 cctctctctc taacgcgcgc acacacacac acacaatcac aattgattat ttgttaatct 64860 tcgttgctag actatgagtt ccttgagggc aggaaccaaa ttcattcatc tctgtatcct 64920 tcatgtcttg tgtggaacgc ctggctaatt tcttagaact gtgccatcca gtatggcatg 64980 tgtcagttaa aataaaattt aaaatgcggt tcctcagtca tgctggccac acttcaagag 65040 ctcaatagcc acagggcgga taatgaatat gtctctgcag aaagctctat tgaacagtgt 65100 tgtcttagag gataccagag gcatacaaca cccaagatgg agggatgatg gatctgggga 65160 gtccttagct tcccagatta tatgtaggtc aatgcctggc acctgcttga aggggagctc 65220 cctgtaactg ggtctggagt ccagtcatat ggattctggg cttggtgact acttctagtg 65280 cagacacact gtttagtccc tttggctaca gccccagttt tcagccccct aagggcctcg 65340 gctaggagtg tactctttaa ctgagaaggt ttcttttcat taaatgggac aggcctttca 65400 ggacaagctc tgatgttcaa atcttgataa ggagaagcct tatcaataag cctgaattga 65460 gtggtagctt tggagtcaga ccaatggctt cattcctctg agcctccttt tctgcatgca 65520 agatgggaag cataaggttc ctactccaca gggctagcat gaagattaaa tatgataata 65580 tatgtaaatg ctgagcacac ctggcaatta gctccttctc aatataaaag ctaaggtttt 65640 tatattgttt tatcaaacag caaagtatat agtctaagag cttctctgga aagagactat 65700 gtgttattca agtttgaacc ctagaactaa gcacaaggct ggcataatag taggcactca 65760 atgaatgttt attatattta atccaatcct attgaattga agacaaaggg aatgatacca 65820 tctgtaatga gaggcatttc aggcagtctg ataggtagtg agggagggat gtgggggcag 65880 tagggcttta tggaagcctt catgagacca taaagggcct tcacagaatg caggctgtgt 65940 tctccaggag agaggctggg aggggtgtca gtccgcggga aggcagaagc tcatgagcat 66000 ccgtgcctga gttggtggag tctgtgggta cagattctgt gtgttcccca gtcgagcaag 66060 ccctcctgcc aagcagggcc aggacatgcc atgggcaggg gaccccacaa ccccattgga 66120 gccccgtggc cctccgtgca gaagctggca cctggcactg gcggctggtg ctggtgtggg 66180 aggctgggcc ctgccgccta tggaaggccc aagggcatct cctcactgct cggcaggtgc 66240 cagcctgctc cctgctggaa tccttgcctt cccaggcagg ctctcggtca gggccgttgg 66300 tccctccagg caatcaacgg ctcccacgtg gaatattttc cccacagaat aaccatttcc 66360 atagtggctg cttttttttt ttttaacttt taatatcctc ccccttccac cctccattct 66420 gtactccaac cagagactga gatgtccccc tccccaccca catcttttct ctattctctc 66480 ccttcctgtg ttcaactgaa agtcagctat gtagccaagt aggggtgggg ggttcagaga 66540 tgaaaaccag ccctggggtc ggccaggagc aaacaatcag taatcctgtg tcaaatgaac 66600 tgtctttgtc ctcaaggtat ctcacctccc agcctctttc ttcctctcaa acgcagagtt 66660 aggaaatgag aacaggagca ggcctagccc acagcttttg aggatttttt agtttgggag 66720 cagagccgac ctcatgggcc tgcgacctgt gtggttgcca gggccctgat agcagaaggg 66780 gcctgtacct ggtttagtgt tctattgcca tcttgaagtt ttttgtttgt ttgttttgga 66840 aatagggtct cactttgttg cccaggctgg agtgaagtgg tgtgaacaca gctcactgca 66900 gcctcgactt ctgggatcaa aagatcctcc tgccgcagcc tcctgagtag ctgggactac 66960 tggagcgtgc caccatgcct ggctaacttt taaatttttt tctagagagg gtgtcttgct 67020 attttgtcca ggctggtctc aaactcctga gctcaagcaa tcctcccgcc tgagcctccc 67080 aaagtgctgg gattatagac atgagccact gtgcccagct gagattcttg gtaaacacag 67140 gcttttcatc cccattatgt agccactcct attcaggagg gtcttagtga tgggcagctt 67200 caaggagaga gggaggcaac caggctggtg tgggagtttg ggcagagaag cccactaaat 67260 tcagcaacct gagtttgtaa gtcactggaa gaaagctggc taggggctcc ccttctctga 67320 gctttcagca tctgaaacag aacctttgcc ctcctttcct actctgcaaa accctgatag 67380 gctggggagg gaaagggata cttcaggtac taggactggc tggagatact ttcttaggat 67440 gagaccatgt catggaagag actctggagg catcatcttc ctcttcttgg atgagaagtg 67500 gaattcctct ctggatgtgc cccagccctg tggagcctac ttcattcagg cccacctttc 67560 ctcatgaatt ctgggtttca gtctctctct gctcctaaag ccctctgtcc taccattgcc 67620 atgtcttctc atgtaatgtg gctcagagct agggagggga cccaagtcgt ggctgacctg 67680 aatggaacag aagaggagga cacctgccct ctttcccttg ctttcctcgc ctcccttagg 67740 gcacactgac ctttgaattt aaagtcagcc tgcactcagg gagcgggtac tatggcaaag 67800 ccctgagcat ggagctttgc acgagtgatt gcttttaaat gaatatttga tgacatccat 67860 gggtcagcta gaggcccact cagacccctg aatctgtccc ctttatcttg tatttgacta 67920 agatctggta ggatataaga tggattagag aaataaatcc cagaaagggt ccactagatt 67980 gatttagtcc tctgtgggct gtaagggccc ctggtttcca aatagagata tgcttcttgc 68040 atatccatat cttacacaaa gaactgtgag tctctctgtc attccttcgc ttttggggaa 68100 ctagactggt agagtgaaaa gaagctctga gttgttttgt tttgtttttg tttttgcttt 68160 tgtttttttt ttttgagaca gagttttgct ctcgttgccc aggctggagt gcaatggcgc 68220 gatctcggct cactgcaacc tccgcaacca gggttcaagt gattctccta ccgcagcctc 68280 ccaagtagct gggattatag gcgtgtgcca ccacgcccgt ctaattttgt atttttagta 68340 gagatggggt ttccccatgt tggtcaggat ggtcttgaac tcccgaactc aggtgatctg 68400 cccacctcag cctcccaaag tgctgggatt acaggcgtga gccaccacac cctgcgaagc 68460 tctgagtttg agtcccagcc ctaccactgc ctttgggcat ttctctttat ctctgtgggc 68520 ttcaggctcc tcatatgaga cgagtgaggc tggaaggctc cttcaggctc tgacacagtg 68580 tgattcttag tgtacgctat tgtcctgggt cgctgtcagt gggtcacttc ttgctgtcta 68640 acctaaagct cttctgctac gcttgctccc tgttaggtcc tcatctcatt tcagtcaatt 68700 cagaccagga ccctatgtcc ccatctccct gggatgtcct atgacctggc gactttcaca 68760 ccacatgccc cttgggactt ggagggaagg gtccccttcc ttccctccct gcttcagcct 68820 cagcccttag ctgggggagt atgagaagca gtttggccct cgacaggtta agctgtttcc 68880 ccagagccta agcaagcccg aactggcctg tgaggtccct gctaatctgc cccattactg 68940 caggtaagaa ttataatcag aaatcacttg aggggccagg tccacgtggg agaacgagag 69000 gcgctgctgg gggttgttga ttctgttcca gagcttaatt ggtataattg ctctcttcag 69060 cctgccaagc cagagtccat caggattggc tctcccaccc tctacgggga gctgcttaaa 69120 aaggtctttt agtcccaagt ggaggattct gctggcggct cggagactcc ttcagcaatc 69180 caatacccct ttcctggctg ccagatggca ggggagccta ggacccccct accctagagg 69240 actagagatc ccaggggagc ctgaaccctg acttgaaagg gtgggggaat tctccagaga 69300 gcaccttatc ctaccgggag ctgggtgggg agatagtaag aggggttgct ctatagccag 69360 gtgtcgaccc ttaggtgttc ccaagagtca gaggttcctg ggggcaggga gcaggtgtcc 69420 aaccagaaaa tacaggggag gctgggcgcg gtggctcaca cctgtaatcc cagcactttg 69480 ggaggctgag gcgggtggat cacctgaggt caggagttcg agaccagcct ggccaacatg 69540 gtgaaaccct gtctctacga aaatacaaaa aaattagccg ggcttggtgg caggtgcctg 69600 caatcccagc tactccagag gctgagggag aagaatcgct tgaacccggg aggcagaggt 69660 ttcagtgagc tgggattgca ccatggcact ccagcctggg caataagagt gaaactctgt 69720 ctcaaaaaaa aaaaaaaaaa aaaagagaga ggggagagag gagaaagttt aatagaagcc 69780 aatactgcgc ctgtctcctg ctgcctcctc cactcccctc tgtgcttcgt ggagtctccc 69840 tctaaggggg tgtttgtcct cactgtctct ggggtgggca agtcatcaaa ccatacagct 69900 gaggaaagca acacaaaagg ttaaaggagg gtatttgtgg ctacatctgt gtgatggcaa 69960 agcccacacc ttccacatca caggagcccc ctgggtcaga gctgccagat aaaatacaaa 70020 tgttgcatgg gacttattca aacttatact tgcttatctg aaattcagat ttaactgggc 70080 attgtcccaa atattacaca ggacatattt atgctaaaat tattatttat ctgaaattca 70140 aatttaaatg ggtagcatgt tgttttattt gctatatctg gcagtgctac cctgggtcca 70200 tgccccaccc ccaagcccac ctggcctgag tgtaaacgcc ccgtctctct gctcatccca 70260 aacacaaaca cagtcctggc cgatttcttc catgtgctca ttccaccttc ttcatttgcc 70320 agttttcttc cttttctcct ttggtgtgtc ccagttccca agtaccagga gcagggcagc 70380 aaacacctcc cctctccagc cctcagctgc tctaccatgc cacttaggat atcatgagcc 70440 aggacagaag atagtttctc tctgacagag cggaaagagg cctggaggct cagcccatcc 70500 agcccttccc taagaggctg agaaagtaag gccctgtttt aagccagtgc catgctcggc 70560 ccagcctggc aaaagggccc ttcccaagct ctcctgccta tgggttacac aaaatgcagg 70620 agatggacta accaggactg taggttacat gtgtgagcgt gcacatgcat gtggccagca 70680 aacgtgtgta ctcacacata cgcacataca tagcaaaggc cacttccaag tgccccctga 70740 gctcacagaa taagttttcc ccttgccctg ccctgaccca cttcctgcct gcctcctagc 70800 acctccttat ggtacatggc catcttctca gctcagccac aatctctgcc ctggtgtccc 70860 acctgggcag gacacctagg tgaacaggtg agcaggttct taacctccca tcatgcacct 70920 cagtgccaac acaacacaat ggtgaggcac atgaattttg acgttagaca acttgggttc 70980 agatttaagc cctgcagctt actagttgtg gggccttggg gggcttaatc tctctgtaaa 71040 atggggctaa tcagagctac ctcaatggca tttttgagaa gatacaggac acggtacctg 71100 gtattgtata cctggcatat gacaaggact caataaatgg agacagtgat tattattggt 71160 gtcatggttg tcactgtagg gatgttctct gccctgagct tcagagaagg aggtactgcc 71220 cctcttaggg agtcaggctt cctgggtctg ttaatttacc cattaggaag gcagtagcag 71280 agagcaaggc ccagatctgc gagatgtgtc aggcccctcc ctggccccac atccagtggg 71340 ttgctgggcc ttatcaatgt gacctgtttt tttttgtttt tttttttttg agatggagtt 71400 tcgctcttgt cgcccaggct ggagtgcaat ggtgtgatct cggctcacca caccctccgc 71460 ctcccaggtt caagcaactc tcctgcctca gcctcccgag tagctgggat tacaggcgtg 71520 tgcaaccatg cccggctaat tttgtatttt tagtagagac ggggtttcgc tatgttggtc 71580 aggctggtct cgaactccga catcaggtga tccgcccacc tcagcctccc aaaatgctgg 71640 gattacaggc gtgagctacc gtgcccggtc atcaatttga cctcttaagg atatccaaaa 71700 tctctgttcc cttcccccat ccacactgga cctgcattct cttccctcaa ggccctcatt 71760 catgacaccc ctttccatct ctccttgttg aaatcctatc ctaccttaag aaggccatct 71820 caaatgtgac ttcttccgtg tggtcctaag caggactcag ggcttaggta tctgtctgtg 71880 gtctctggaa aatctgggac taagatgctg ctcccatgga aagggccttt ccagggttag 71940 gaggagagtc tgggcatctg ctgggtgggc atctgcagac gggggaaagc gggaggtgag 72000 agccctcacc acaatagaat ggggtgtcaa agcaggacag ccaggatcct cgaccatccc 72060 tggtaagggg ggctgtccag gtctcctcta gcttactggt gtgcttggtc aggccatcag 72120 tttgaggctt ggaagcagag gattcagagg gcatgtattc actcatttat ttattgccca 72180 tttatttaaa aacattgagt gccatctgca aactaggcat tgcgctgggt gctgcgagtt 72240 cacagctgaa tgtagtgcct gctctggtca tatatgcagg aggttgaggt agagccctca 72300 tctcttggag taatttatga tcaaatctaa gaacgagggc tcccaggacc agctgagcac 72360 taaccacagc aggaaatggc tttctacatc ctcattcagt cttcctctct tctccagcaa 72420 tccaaggttg ttagattgga gcatcagatc tggaaaacct agctcagtgc tttaaaaaca 72480 ttaacgtgct gatgaatcaa tgaatcacct gggagtcagg ttaaaatcca ggttctgatt 72540 tagtaagtct ggggtggggc ctaagattct gcatttctaa caagcttcca ggtgatgctg 72600 gggctgatgg tccatgggag tccacattaa cacaaagctc cccagggtgg ttcagccaca 72660 gaccatcaag ggtgtgacgg ccaccactgg agaaccatgt ccaaggtgac aagccttgcc 72720 agcagagggc actcactgaa gggcttttgg tcccatcatg tcaagtttga tgtgggtggg 72780 aagtctttcc taactacagc ttttgaagag tgtcctggtg aatgtgcttt cacacccaag 72840 actgcagtgt tggcctatcc acccatggcc cccttgctgg tgaactaggg agtgtttccc 72900 agtggtgctt gagggcttca cagagagagc actgatgaga tgctcaggga aaagacagaa 72960 gcactgctgg accttctaca agggggcttg tgctgttggg atgaaagaat atcaccccat 73020 cccaccctac tgatcatatc acttctcctt tgggttcagg attatggagc tgtagggcct 73080 ttgtgggtaa ggctggggtt tccatggctt aaatgccata tggctatgtg ggaggaccat 73140 ggctcagggc taagcctcgc ttcaagatag gaaagtccag caatagatcc atggtacagg 73200 tcagtttgtc tggtccctgt gagaccatct gcttagggca ataggggttc agcccaagtc 73260 ccaacctggg gctcagagac agtagcatgt tgataaaggc tgtcgtggcc tgggcacatg 73320 tgtaaaaagc taaggttgaa aaatctcctt gttttgctgg gctgctaaga tcttctaggg 73380 ctcccataag taatcctcat atcaccctag aatctctggt tatgcacaag caggatcacc 73440 caccatctgt tcacctgttc tgagaggcag ctaatcaaga acttcaactg tggcatgcag 73500 gggcctccta gaggtcatca actgagcaag aagccccagg actttagcag gagccccttg 73560 ccccctgggc ccttgaaggg acaaggaaca ggtggtgcta gggatgggtg acagggaagg 73620 gcttaccctt ttggggaggc atttttcttt ggttcggttc agagggagga catttccctc 73680 tgccaatgct gagaacatct ccattatatg gtcttggact atcatagaac agttagcaag 73740 gtcgaggtca cactcagaga gcaagggtaa tatggcatag atttggaagc ccgggagtat 73800 ggatgtgaag ggaaatactg gctatccaca aacaggctat gggttcagcc ttgggtctga 73860 gaggaagagg tggggtcatt catagaaaca tatggtgtag aggatcaaga aggatcaaga 73920 actgtgggtt ggtgggaggt ggaggttgca gtgagccaag attgtgccac tgcacttcag 73980 cctgggcaac agagcgagac tctgtctccc aacaacaaca aaaaaagaac tatgggttgg 74040 aatggagcca aatatgggaa ctcgaagagg cgggatacag gatacatggt ggccaagtca 74100 caactttagt cccagggagc ctcaaaggcc ctgttgaggg cctggctggc ccagtctagc 74160 cccaaagctt ggtctgcatc ttgccctgga ttgacccctc tccatcctct cttccaccag 74220 gcgcctagat gccaacctca tctccctggt cccggagagg agctttgagg ggctgtcctc 74280 cctccgccac ctctggctgg acgacaatgc actcacggag atccctgtca gggccctcaa 74340 caacctccct gccctgcagg ccatgaccct ggccctcaac cgcatcagcc acatccccga 74400 ctacgcgttc cagaatctca ccagccttgt ggtgctgtga gtgctgctct gttccccatc 74460 cccagtgggg tcctgctggg ggctgggggc tgcatgttta cttgagttgg attggagcct 74520 ggctagcttt gctcttcttt gcatgttgct aaaccttctg gcctcccttt ggaaaagcat 74580 gaggatgacc attccctgtt gatcccatag ggttatcatg aggtcaaatg acatgagagc 74640 ttgaaaagtg ctctgagagt cactaaaggg ccagggtact ataatcagaa aataggaatt 74700 aagccattat tttcttgcca tgcacatata ataactatac cttgataact ggaatataaa 74760 tcaagaattt cttaagtaat ttctaccata tttgacaatt agatgatgca gtcatatgct 74820 ttggcacagg gatgggataa attcatctca tttcccaggc cttaagaagc agagaaaagt 74880 catcacttat aattagacag caaagcccca tcactgtaat tacctgggag atagcatccc 74940 tggcaggaat tggggactgg ggttaactct gcctccccag tgggaacttt agagggcaaa 75000 ttagctccca gctaattgct atgaatgatt ggtaagttca tagggtaatt actaaaaggg 75060 cttggaggtc atgtataatt gctttgggtt acacactaat tgctgcagga acatggctta 75120 ggaacatgca tgttagtgac tctactggat ggtaacattg cccccacgcc ccccgctttt 75180 gaagatctgg cgtgattttc taaaccaggg aatggatgat acctagttcc atttgagaga 75240 tgaaaaggca ggcaccaatt tgtttgtcta attctctgtc ctgggaagct ggagaaccaa 75300 ggtcaatatc cttgtggact atattcttct gtcagggtgc cataccctcc cttcttctgg 75360 gagtcacggg gcagggtggg ggggtattta gagagggtaa acagaacaag gtgattggtt 75420 ctaactttgg tgacatccct gtgacattgt gagagggtgg ggtcaggcct ctgtgaggga 75480 taagattact ggagaaataa gaacttcatg gttggcccca ggtggataag aaggagcagg 75540 catttattga gcccccatgt ggtgtactga aaactcaaaa aactgtgaga tatagccact 75600 gcccttgagg ggctttcagt ccaattgaaa gagcagaata aatatgggaa aaaaataaca 75660 aatgatgtag tgaaacttat gctaacacag ccagatgata aattcagact catagcagca 75720 tagatatggt gggaaggctg aggtggaggg gaaggaggct tcctggggga ggtggggtgg 75780 gacctgggcc tggaaagatg atgaaatgaa gagaatgggg acattccaag cagatagcaa 75840 taaagacatg gaggggagag taagcgtgtt tggctgccag tgaccaggcc agtgtggctg 75900 gagcagaggg tccccatgca gagctcagga gaagaagctg gaaaggtgca tgtcgggact 75960 ttcaacgtca gaacagagag cttggcttct ctctgctcgt gtgggaggga agcttgaagg 76020 ttttcaggca agggttaaaa gggagtattt taggaagatt aatttgaagg ctggattgga 76080 accgaggcag actggagaca ggaaggtcag ttcagaggtt cttggtatca aagtccagta 76140 gtttaaggag gtggcagaag ggatggaagg gaaggcatga ctgtgagggc tgttttgaag 76200 gaagaattga cctgattgag agattttcag agggtagaag accaagaaga aggggagtca 76260 tgggtgactc caaggtttgg gtgggagcca aggaattggc ctataagtca ggcctgttat 76320 agatctagga gagattgagg ggaaggccca ctgaggacaa ggagggacaa gggactaagg 76380 aaagaaaacc tactggtgcg ggagcaaagc tgatagagaa aaactggtcc ctttgcatgg 76440 ggagaggtta gaaacattac cccttcatcc ctcacctgcc tccaactcat gggatggagg 76500 agagaggctt ctagcaggag gtgacctgtg aagtggacag gaggctgggg aatgctgtcc 76560 agggactaca ggatgagctg taggaaggcc caagaggaag gtaaggaaga ctggatatga 76620 tgggaagcaa aaaagatatt tggttaaaat caagggttgt acaggggcct ggctggaggt 76680 gtgagcaggc aagcagggtg gaggagaagg ggcttctctt caaccccaac cctgagcttt 76740 catcatcggg ctgctctgcc acatgtggaa tctccctcac cactgctctg gcaccaacag 76800 gacccttagg tagcacgtag ctggacagca gtggtgtcat gtgtgtaatc ttgtctcttt 76860 atctggacta tatacctcaa cacctctccc agactggacc atttggggtg cccaagacac 76920 cagggttgaa tgagtggttt aggggaggga attggaggcc tggagctgga tgtttagctg 76980 agatggcctg gacctgagat actgggggag gcagaagggg gtggaaaatg gagacaggag 77040 ttcaacagca ggtaagagga atgatagagt ttgatgacaa ttggtttata ggtgtgaagg 77100 aaatgggaaa aattaaaact cctcagtaag gggagatcac tcccagaagg cagtgttttc 77160 ccctttggat cagaagctcc ctggggacag gagtctgtct cctgaaacgg aacttcatgg 77220 agcaggtggg taaggatggg gaagacagac agaagcttta aatccccagg ctcgcaggag 77280 tcccgtcctt acccatctat gttgggtgag gcctgcccat tgcctttata tttgaacaac 77340 gtcttggagg ccagcctgat tttcttcccc ttgtcagtga tttccttctt ctgcctgaag 77400 atgtttttaa attcttgaat ttaatagttt aactaggata tgtcacagta tggaatagtc 77460 agcatctaat tatcctggaa caatgtatgc ttttgagctg cagcttccat tctttcatca 77520 tcttagagat ttcttttgtg ttatgtcttt gaatacatct tcagtctcat ttttggattc 77580 tctgctccag aaacatcaat tatgtttgtg ttaaatcacc tttatgtctt cagcatttag 77640 tttttgtttt aacatttttg tctcattcaa ccatagtcac catgattatc tcaggttttc 77700 tctccgacac caattcaatt tttagcagtg tctgcactgt cccttgtttt aaatttattg 77760 actcattttt tcaagtgatg tagtgttggt cctcaactta tttccttaat ctactgtgtc 77820 atttttcctt tcacttaaga acattcatct ttgaaatgtt gttttattgg attgttttgt 77880 agcagaaggc tactgtgata aatttccttg tgttcctggt tcattagtca tcttttcttc 77940 taggcttaaa actttgcctg tcttttgcat cctctttctt ctggctttct gtttccctgt 78000 tgtatgtttt cataatttcc atgtaatgtt gtttttttcc ggcttatgct tggatagctc 78060 tgtccagttg ttcaagctgc atcacttcgg gtcccccttt gcctggaatg cccttccttc 78120 tgcttgttca gttcacctgt ccttcaaggc cctgctctgg atcaccacct ctataaagcc 78180 tgcctcgatt cctctggcct caccaatctg cctgttctct gagtgtgtct gtagctctct 78240 ccagttgtaa cagaaagctt cgcctttaat tatatgttga ctgcagtggt ttgctgttgg 78300 ctcccataag tacgtctggc cactgagtca ggcagactgg gtcctgggga gcccagccat 78360 agaagaagcc tcagacttga gcccctctga catcagacct gtcttctaga gcttaggggg 78420 aacttgcttt agccttggcc tgaggctctc ctctcccttt ttcctttaat aggaaatcaa 78480 tatggggtag cctgtggtca atccagcctt ggagattgtg gcttcagaag tcagtcttgg 78540 ccagtcagtt tgttcctgga aacaaacgga tggaccatta gggtcattct gtccatgctt 78600 ctgcctacac tttccccatg ccaccatgtg ctcttccatc ccctggcccc agtgccgtgg 78660 gcacccattc tgatgcgtct ttccttcccg tgcaggcatt tgcataacaa ccgcatccag 78720 catctgggga cccacagctt cgaggggctg cacaatctgg agacactgtg agttttgagg 78780 tcttggtcaa actctgtcct gcctggtgat gaaagcctgg agtcttggag tggagggagc 78840 acacagaggg aagagccagc agtcctggga tgctggggtc tggccccggg cttgtttacc 78900 cacatgcccc tccctggcac tttctctgga atatcactga gaatgaattt cttcctcctt 78960 caggtggtca gaatgtgccc tctgggagtt cagaatggcc cagggagtag gaacctggga 79020 tctccctgtg ggcagagacc accaaaataa tgaaaattct ggacagaaat gggaactgca 79080 agaagctcaa ggaattaaaa tatgaagccc ggggaataga aggcatgggg gtggggctgg 79140 ggctgacttg atggcagttt tgacatctgt gtaagggtgg ctgctgacag aaggaagagt 79200 aatactgcag agtaatgctg ggactgtttg ccaattgatc gaactagcta aatgattaat 79260 ggggagatgg cccattgagc actctagcct tactataaga agcacccaag ctgttgactc 79320 tcaaaaagcc atattattca cacatcatca tcctggaatt cagttaaggt cagaatacac 79380 ccaaaccctc ccatcatttc ctgtggagtc ctgccagtcc ccacacagtg ggctgagtgg 79440 gccccacttg agcaaacaac tggctgggct ctccaagtgt ggccgtttct gccagctggg 79500 gtccctcagc atccatgagg gagctggctg ccatggccac ctggtacagg cctcggagag 79560 ggtcgatggg gagagaagga cccccaggtc accaagtatg agccactgag gttggggtag 79620 agctcatggt gtcaggggga tgctcacagt ttctcttacc ccccacttgc tccaccttgc 79680 cctccataac caacccccct tattctccac cccctcccac atatacccca ggacatcaat 79740 cctgaggtga ggggttttca agaatccgaa gcagcagagg agaaaatgct gagtcaagct 79800 gtgatctggg aagcaatgta ctggggaaaa ccatttggga aaacttatcc caggaggcct 79860 gggctaatgg catttgtggt ggtgcaggga aaagtaatag ggaggctgag acccaagctc 79920 cactcttttt tcctgtgggc acaagaccca gaagtgtggc tgctttcttg ggcttagcct 79980 gaagcgggag aaatggcaga gccagcccag ctttgccggc atgctcaccc tccggaagac 80040 tgaggactga ggtgtggggg acatgcccca gcctggaggc aggaaaattg gaacctcttc 80100 ctgccactgt gcgtgacttg ggctacatgt tcctccctct ctagctgtca tagacagctg 80160 ggcagttaac tctggacagt ctagcccact caatcaacat ctgagaaaat tgatgtccgg 80220 ggaggtgtca tggcctgccc agagttactc cctcagttag agacagagca gatctagggt 80280 gcattaatga gaaaacatac atgaaagtcc ctgggggaga gcctgctgtc ctcttttgtc 80340 tgggtcaaat gtgaactgtg tgagcctgct atgtgccagc tgtttggggg aatcctagat 80400 gtaagagatg gtgctcatta ccctgcaaaa gcctgtgtgc ccacagccac cttccctcag 80460 gagatataag agcttttagg gcagtgtttg gagttgccat ggatgagcag ggcaggaggg 80520 agtccaatgc catgaaccct ggagtgtggg cgctgttagc tttgctggga aatcaggtgg 80580 ggagggggta catacactga ctatgtgttg gcgcctctaa aaaaccaact agcagctgtt 80640 tcttcacttt tggggcacac tgcctaattc tttctcaatt agccactgga ttgccgcgca 80700 gggcatttgg ctcagctgat gctggctgaa ttacagagca atcctgccgc taagcgccca 80760 ctgtaccttt catctacagg agcacttgat tccatcacac aaagcccagg gcctgcaccc 80820 actttaccaa tgggaaagcc aaggctagga gcctggtggc aaagcccttg gcatcagaca 80880 gcctggggta cagtgccact tctaccagct gtgtgacctt ggtaaaatca tttgcctctg 80940 tgagtctccg ttttctaatc tgaaacataa tgccagtaat agttaactaa tcttacaggg 81000 ttggcctgtg gattaagtga gatgatgtgt gtaaaattct tacaaaccct gacaagctgt 81060 ataggagcct gccctacact cctttctcct ttcccatctc ctcctctgag ggcctccatc 81120 tcaacaatac ccccttcttc ctgcccttga ggggtgagtt cttcttccaa cttttgatct 81180 ccaattaaaa aggggaaaag tgtgttcttt atcctctgtt ccccagagca tttgctgggg 81240 agcctgcagg ggcagccaga tgtggcaggg ggaggagtag atgggagaat aaaaggagaa 81300 agaaatgata gtcaaacctt ttaattgttg cataattggc acgctttgca gtctctggag 81360 cttttcatgt gagcttctca ctggaagggt atttctccct caggccccat aaatctctgc 81420 cacttagact tctctcatct ccggcagcag agggaagatc aaacccctgc ggcccaagct 81480 gcggtttggc tggagctgtg gtttggctcc tctgcagact cctctaagtg gggaaagcgg 81540 cccccagcca taccacctgg ggaggagggg ccaggtgcgg gcatgcacac acgtggtcac 81600 acaggcatct ggaactgcac agccaatcat gtgcacgtgc agcacacgtg ccacatgttt 81660 caagccctac atgaccctca ctccacgtag gcagccccac cccacaaccc agcacagcac 81720 agaaatgccc atcacaaatg cacctgcagc aacacaccct ctgttatgcc ctcagctctg 81780 gagaccaggc tgtttggctt ccaaccctgg ctttgcccct gcaagctggg tcagccagag 81840 gaagtaactt cacctctgtg agcctcactt ccttcatctg cacaatgggc tacctcagag 81900 atggggagaa agtgtgagag acatacacca tgctctcttt tactccaaaa gagccagcaa 81960 atccaagtca tttgggcagg gttcagggga tgggcactgt taagacctct cttgtttagc 82020 agaacaaatc gtgccaactc cacccgcccc acccccttcc tacagaaacc ttgttgccct 82080 ctgggaaagt ctgacctcgt aggaaggaag ctcccaggac atttctcctg atgtagtcat 82140 catcattcca gactttgcca agaaacctca gccagaagca gacaggtcat gctccatcct 82200 gcatccacaa ctcctttcta cttccacaat gaggatgacc cgaacttgct gggaaattga 82260 gaatcagagg tggtctagtc cccagactgt gaagtagtat ttccacttaa ccagatcaga 82320 ccaaatgtag gtatttgggg gtgagttgcc catgtcactg taatttgcaa gtgacggtaa 82380 accactagaa accatacctc ttgctttgta atacgttgct tcacttagca ctagctccgt 82440 caaggtcaga tcaatcatag gacttcccag gtatctaggc tgagagataa gagagcatgg 82500 tgaagaacat ttctaggaag gttttcccag gaggtctcag gacctgaaac cctaggtcag 82560 tgtttggaaa tgtgtgcctg gaggccatga cgagctagtc tagcctgaca gctaatgatc 82620 tttgaaaatc ttcgcaagaa aaaaaaaggt cgagcaaaca caattgcatc ctaaagggcc 82680 caccctcatg ctctagatct gggaacttga tggctgaatc tcagctagaa cccaggaggg 82740 gacagggccc aggatggacc ccggggacaa tagtgtctca cctctcttgg gcctgggtga 82800 tgctccttgg gccttgttat aacctcacta aacaaatcag gcccaactgg agccctgtgc 82860 tggctggaca cttccctttg gaggagagcc tggggccata gtgagaacag aaatcttctg 82920 gccccagctc tctctctaaa tcaggaccag cattcaacag gggcctggga caggaagagg 82980 gcaaagaaga aatgtattga attctgcctt ataatacaaa attgtacctt ccacatcaaa 83040 tctccttctg gtgatggatg ctctggtgat ctgtttccac agaacatttt tacttcaaca 83100 gcaactgtag ctagaaaatg ttcctctctg tgtgtgtctt tgaaaccatt aatgccaaca 83160 ggaaactggc tctgacttgg ttctgagagt atctggccac cccttactcc tggccctgat 83220 gtcagccttt ggccagggac aggaaaccag gtggacttcc atcgaggtcc tcccagggct 83280 cagggttctt cccagggtag aatgaggatt catcttgact tagcagactt agattcaaat 83340 cctggctttg ccaccagcat gtgacaggtg caagtcacct gacttctcaa gagcctcgat 83400 ttccacagat agaaaatggg gataatagtt ttcaattgag aagattgttg gcaaggatcg 83460 cattaaatga gatgacttca cgtgatgcct ggaacattat aggtgcttga tttatgtcgg 83520 cttcctttcc tgtcctccca tttaatgttg gatggaatgt gtcctaaggt ggccgggagg 83580 atgttaccca gaagtgtcat ttcacagtgg tccatgctgt gtctcatctc ctctgtagga 83640 tggatgggaa tttccaggca gtcaaggcat gactactgtg ttaatttcct gtggctgcca 83700 taacaaatta tcacaaattt agtggcttaa aaaaacagca acttattctc tcacagttct 83760 agaggtcaga agtccaaaat caaggtgtta gcagggatgg tgccttctgg agggtttgag 83820 gcggaatcta ttccatgcct ctcacctcac ttctggtgtc tggtggcaat ccttgacttg 83880 tagacccatc actccaatct cttactctat cttcacgttg ccttttttgt tttcttctgt 83940 gtctctcctc tgtgtgtctc ctcttcttgt aagaacattt gccattggat ttggggccca 84000 ccctgttaac caaggatgat ctcatcttga gatttttaat tccttctgca gaggccctct 84060 ttccaaataa gctcacattc acaggtacca ggtttggaca tatcttttgg gggctacata 84120 taattcaacc cactctacag ctatcctggc gggctccatg acagagccct ctggggatag 84180 gctgataggt tggggcaggg gtccagatga atctcgcaaa tttgccttca tatctcaagg 84240 taagtgctga tcatcccact agtcactggg caaataattc cctgcagtta agagaattag 84300 aaatattttt tttctgaaag gctgtcttgt ctcatatctt atttatcccc caatcacttg 84360 ttgagaaaga caaggcagga attgtgtatc ctcatgttac agatggggaa actgagacag 84420 aaggtggatg acttgcccaa ggacacccag ctaattaaga gccaggattt taatttgggt 84480 ttctctgcct ttggcccagc gtcagcccat cagaccacac tgcctcttga ggctaagaat 84540 caaggaactc tctttccttg ccagttctta atcatcttac atttgtgggt ttattagctg 84600 caaatttgta gggttttgtg gcttatagag gaatttattg ctccatataa aaattttttt 84660 ttctggcttc ccccgcagaa ccactaatca ctttaaacag gcagaaaatt gagctgccca 84720 atgagtttgt gttgcgttct gtgaccgtga cgagttatta atagttcttc ccagcacaac 84780 attccttttc ccattgccct ggtgtggggg gtagccaagg tcactgctcc tgtcattgct 84840 gcctctccct cccctgctgg caatggggac aaaacttcca ctgtgaactc taccccctct 84900 gccgactgct ccaggcaccc cagccagctg gtctggaatg gagaggaaga gaggttaagg 84960 ggggtgggaa gcttggccca caggttccgt agtagatgta atggagagga aacaaagttc 85020 aggtctggcc ctcccatatc tgtaggaagg aggagctggt aagaattaaa gattaccact 85080 ctcttttctt ctgtttcttt atattcactc attccataaa tatttattaa gtttttagta 85140 tatgccaggc caccattata ggcactggta aaacatttat gaacaacaaa gtgtagtccc 85200 tgctatcaca gagactaaag ggttctagat cagaagtcct gagttctttt aaacttaaca 85260 tgaatgcact gttaccagca tttctgacct catcacccta accccttaac cttggcatgt 85320 cccaaggctc aagttctccc aagggctcaa gttctgttta cgcttctctc cctccatgtt 85380 ctcatctggt cttgtatgtt tgctgattac tccagcccag acctatctcc tgaactccag 85440 acttaaatat tcaaaggcct tcttaacatg ctcactggat atctaacaaa tatccagact 85500 tccagactaa accccggagt ctttccctga acccatccca ccagaagtct tccccagctc 85560 agtgatggca gctttactcc tgcagttact caggcccaaa ccttggagtc atccaaaaca 85620 actctctttc tctcacatcc cacatccaag ctgtcagcaa actccattgg ccccatcttc 85680 caactatatt aagaatctga ccacgtctca ccccttccac tgctaccacg atgctccaag 85740 ccgccatcct tctctcacct gggttattgc agtggccttc tatctggtca ctctgctttc 85800 attcttgtca tcctacaatc tgttttcaac acaacaacca gaatgactat tttaatatac 85860 aaatcaggtc atgttacttt tctgttccaa accctccagt gattcctcat catactccag 85920 aaaaaaaaaa gccaacattc tgtactggtc tacaaggcct cagatgctgt ggccccttgc 85980 cgcctctctg gtgtcacccc ctcctcaatc tagtccaggc acgatggcct ccttgaatat 86040 actacttttg ttcctgcatc aagccttttc cttttgtctg gaatgttctt tccaccatta 86100 tccacatggt ttacttcctc acctcctcca ggactttgct caaatgtcac cttttaagta 86160 tcctccctgg tcaccacttt gcaacctcca ttcccagcac tctccatttt ctacttaatt 86220 tttttccata gcatgcaccg caatctgata tcctaataat gtatttactc acttaattgc 86280 ctgcccctta ccgctccatg agttttgttc acttttgtct ttgcagcact tgacccagtg 86340 ccaggcacag ggaaggtact caataaatgt ttggtgaaca aatatttcaa caaatagatt 86400 tatggctcct ggcaaatctt tatcctctct gggcttcagt ttccccatct gtactgggag 86460 gcattggacc gtctaacatc taaggttgtt cccagctgca gtcacatcta tactccgaga 86520 tatgcagtct ctagcccaga cctctcctct gcgccctaga tgtaagtgtc ctctagtcag 86580 tcctatcaca gcccaggagc tggcacaggt gtcccatgct gtcttggaga aatggcctgg 86640 atatggactg aacccacgag gatggtccct tggccctccc tttggtccag gtcaccttcc 86700 cataagccag ttgttcagaa ctgcaagacc ccagcttttg accaacaggt ccaagtctct 86760 gggaaagatt atcgggccac attctgctgt ggtcaggaac acctcagagc cttcaggtcg 86820 cctgcattaa cgggccccaa acatccaggt gagagggatg ttgcctacag aggtctgatt 86880 gcatccagag gtctgattgc atccagaggt ctgatggatc ctgttctggg tgccatatct 86940 taagcgtggc aaaggctctc ctggggaggg tggtgagagt atgaagtgtc acaaaatgtg 87000 attcaaagaa ctgaagacaa gccttggata gagtacaatg atcatctttg gaaggcagat 87060 tacattttat gctcttccag ctgacaggat gagggccaag cagggaagtg aagacgggca 87120 ctccagctga cagtaaggaa ggattacgta atctgcagag tggcccaaca gtgacagcac 87180 cagacaccca gcatgtcagt gagtgccttg ccctttagat agttctagta caaactggat 87240 ccccaattgg gccggaatct cagagatgat tcctctttgg gggcaggttg gactattact 87300 ttttaccacc cttctaattc taggtttctg tggttccagg tgcttcatta gggcagggca 87360 acaagtaatc tcaatctcag agatcagtca ttttcattaa aatcagcact gctattgctt 87420 caggtagatt ctgaattttc tctctggcaa gaaagagaac gtaggtggaa ggagggaggt 87480 tgtatctgtg tctctcaatg gaagctaatg acagctttac tcattgctgc atttattaat 87540 gggtggcctt ttatgtagat tgtttagaag gtaaaacaat actattagtt ttaaagtttt 87600 ggttggttcc tggctcaccc agtcatctgc cccaagcacg tggagtcata tttaggacag 87660 aggctctgtc tgcagtatga gaaaagcaat taagacatgt attcattgtg ctagcttgcc 87720 tttgttccag gcctttggga gataaaaggc aattcagagt ttacaatctc tccaaaattt 87780 ctgtgttttg catttcacac tggggagcta ggctcaccca tattccctca tgtaccctta 87840 tctgtgatcc agtaataaac acaaatgtta gccttgagaa taaaaatgta agtggaaacc 87900 caattaatga tattcatctt tcttaattgc tatgaaaagg ggatgggcca acttgcatgc 87960 tgacttttgt agtatctaaa gaatgaaagt gtctgttcta gtagcttatg tctagtaaaa 88020 ggaagaggtc aaaggaattg gcattatttg aacatctact ctgtgctagt ctctggacca 88080 ggtgaacaga gcaatcttat gcaatgggta tgtcccattt acaggtgaag aaatgagcta 88140 agaaaattaa gagatttgtt caagatgatc ctgttggtgc acagaaggca agaactctct 88200 ctaacacaga taacatacaa aaagacatct aacttccaaa tcataattat ttggctttga 88260 catgcttata agcatcaaaa gaaactcctg cagaagccca atcctccacg tccatttatc 88320 ctctctccct aaaatttttt tgttagccct gctatttaga gcctagccag gatccaactc 88380 tgtgtagaat ataggaaagt gttttaaacc aggagccaag aggcctgagt ttgagtcctg 88440 gctctgggac ttgcagcaat ttacctcctc tctccaagct catttcctca tcaataaaaa 88500 aaaggggggc caggcgcggt ggctcacgcc tgtaatccca gcactttggg aggctgaggt 88560 gggcggatcg cctgaggtca ggagttcaag accagcctga ccaacatgga gaaaccccat 88620 ctctactaaa aatacaaggg tgtggtggtg catgcctgta atcccagctg cttgggaggc 88680 tgaggcagga gaatcacttg aacccaggag gtggaggttg tagtgagctg agatcgcgcc 88740 attgccctcc agcctgggca acaggagcaa aactctgtct caaaaaataa aaaataaaaa 88800 gcaaaagaag ggactggaca ggatgactgt aaaatcctgc cagctctgtt gtctgaatct 88860 acagctgaat ctacagggag gctcctcaga gatctctgaa gttggccatt tctgcacagc 88920 ctgatggaaa agagaaaatt aggcatgtgg gtccttgaac agcttagggc tggggttgcc 88980 ttctcagcta cttttaccga ttgcatggat ttgccatttg ttttgattag atttggctag 89040 aaacagttga gcagatgaag gggtgtctta gctatggaca gctgcagctt ccagactgag 89100 ctctggctgg agttggaagt ggtttctctt taacttcagt ccttccccct ctgcttctag 89160 ctcccgggag caggagacag aacaatgaac tcttcctggg cttctggctg attgtgggct 89220 gttaaagata atagtctatt aagccatgga tgagttttcc tgctgaagtt tagggcatca 89280 taagccatcc tgcaatgaaa gatacacact gagcctggtc tgcatgttag agtctaatcc 89340 caaaggccaa gacactgcca tgaagcagcc agctaccaag gccaaaggag cccccatctt 89400 gagaccctgc acagtggact caaatgatca tctacaacaa tgaacattca catatgtggc 89460 cttgtgcctg tgctgtggga gatccaaaag tgaacccata tgcttcccaa cctcagcaag 89520 cttcctgtct cattggggag acaatatgca tgcagaaaac ccaactctgg actcagattg 89580 cctggacttc aatccccgcc cttctaatac tagctgtgga actgtggtca agtgattatc 89640 tccctctgtg cctcagtttc tcatctccaa aatggaggcc attatggtac ctacctcaaa 89700 gggttggcat ggcaattaaa tgaattaata tatgcaaatc actagaacca tgactattac 89760 atagtaatta ctattatcct ctattattac cattgtaatg ctatgcatgc agaggacgaa 89820 aatcccagaa aagttccaag agagtgagtg aatcattctg actacaggga ttgggttgga 89880 ttgagggaga aaggtatagt tggaccggac ttcaaaggat gactaggatt ttaacagctg 89940 aggatgtggg gaaagggtat ttcaggctga gggcatgagg ggcgcaagta actacacaag 90000 ggtgtgaaaa tggcacaacc cattaagggc tgaaatggca gatgaaactg agggattagt 90060 taagggcatc ggagcccatt aagggatttg gattttgtct gtaagccctt gttctggaag 90120 cctttccaga tttttcaaca ggagagtgac agactcagtt ttgcatttta aaaagatctt 90180 tccggcagat gtgtagtagc taagagtgtg ggcttcagac tcacactgcc agggttcaaa 90240 tcctggctgt gacaccgaat tagttcaggt aggctaaatg gctgtgacat atagactgaa 90300 acatgtataa tggcacaaac acaatagaag tttatttctg gatcatgtaa cagtctaagg 90360 agagtgtgtc ttcttggcag gtggcctcat ccatgtgatg actcagggat ctggcttctt 90420 gggagcttgc cacctccagg gcctcattgt ttttggcctg tagctggtgg aagggtagag 90480 agaccagtga ggagacaagt tgtctatttt ataaatcctg gactagatac agtatgcacc 90540 acttctgaca cacctactgt gaaggaggct gagaagtcca cttgctccat tcctggcatt 90600 cttcctatta tgataagcca gcactgccca gtagaacttt ctgggttgac ggaaatgttc 90660 tgcatctgtt cttatatgat agttgcttgc cacatgttgc tactgagcac ttgaagtgtg 90720 gctagtgaaa ctgagagcca aatcttacat tttatttatt tgtagttaat ttaaatagcc 90780 acacatgcct agtggctatt gtattggtca gtgcagctat ggagaaaaga gagagtagat 90840 ttttacttac agacagtaga ctctgccaca gccaggtatc agttgtgtga tatgagtgac 90900 taatttaatc tctctgtgcc tcgctttcct caactgtcaa atgggtataa tagcagtaac 90960 tgcctcttga ggctgttggg aggattaaga gattattcag tcaaaacact tagtggctgg 91020 cacacaggtg ctcaaccaat gttatcatat ggatgacttg gaggcaggga cagcagataa 91080 gaagtgtata gcagtccaga ggagacagcc ctctagaatt acctccaagt attgcccttt 91140 gctccaacca agcgatattg cttgctcttt tccaaacacg actccacagc actcacttcc 91200 tctaagtcat gctctctctt cttgatccat tcatcccttc atattgaaat cttgctgttc 91260 tatgacacat gtcacatttc catgaagccc tcatggtctt tccagctctg tgtgatcact 91320 gcagtctgtg aaggtcctga tgcttcgtgt gctctccaca gtcatgtcaa agatgctctt 91380 tcattatctt gtgcatttgt ctagatagga gtgggttttt ttttttaaac ccatctctct 91440 accttccact ttacccagca gaattctttg tgtattgcaa ggaggcaaca aatgttcatt 91500 gcactgagtt gaatccatca ggagcggaga gagttgcaaa gacatgttcc ctggacaatt 91560 agagccacag attacatcta catggtacta tatgaagcag gactttggca atttcccagg 91620 tcataaaatc tgtgtagacg tccaaatggg aaaactccgg ccacctcatt ggcatggagt 91680 ttgcagcatt ctctttgacc tttgcacagt ttttattctt tcccatttct cgacaaaaat 91740 agacttgtag ttcacatgcc ttttaccata tgtcttttgc tactgtctgt taggccactc 91800 ctgcaaaatt cctgaccaga aactgccaag gagggctgga actactcaat ggattgcctg 91860 gacatgggtc caatgaactg tataaggact caatgatagc aaagtcacat gaaaccatcc 91920 catggtctct gtggccccag gcaagccact caggactatc ttgtgccttt tgcaacatca 91980 acctcaaaat cttaaggacg ttcccccaaa tactgtagat tctcttaaga gtccattagt 92040 gttttcatta atcaatttat ttagatgtct tcattgcttc atctatcaag tatttgttga 92100 gctcctgcta tgcgccagga ttgttctggt gtttagaata tgaaacaatg tttctatttt 92160 caagaagctt tcattctaat tgggtgagaa gaaaataaac aaatatatgt caggtgacta 92220 agttctgtga agaaagaacg gaagctgtct cattctatat tgctacataa caaatcaccc 92280 caaaatttag tggcttaaaa tatcacttta ttattcttca tgaattatgg ctttgctggc 92340 tcatcaggtt ggttcttctg ctggagccat cccctgggct cattcatgag gttgtagtca 92400 tctggacgct caactaggtc taggaggtct agggaggcca ctcatatgtc tggggccttg 92460 gtattggttg tcggctggtc ttctgtatcc acatggccct catcattcaa gagtctagtc 92520 tggaatttct tacatagtgg tgggagcatt ccaagaaagc aaaggcagag gctgtgaggc 92580 ctcatgtgat tagggaccaa gtcacactat atcacttcca tttcattctg ttggaccatg 92640 ccagtcacaa ggccatccca gcccaaggta gatgtcacat cttgagagga gaagctgcaa 92700 aggatttgtg atcactttta atctaccaca ctggataagg gaatagcaaa tgagaggata 92760 tgctatttta gatagagtag tcagagaagg gctctctgat gaggtataat ttgggcagag 92820 acctgaatga agtgagtaaa ccatagaagt atttggggaa gagcatgcta ggcctatgga 92880 atagcaagca aagtctctga agccagatca atgcaatact ttgcgtttga gataataatg 92940 caatattaag acttttcagc tgggcacggt ggctcatgcc tgtaatccca gcactttggg 93000 tggccgaggt gggcagatca cctgaggtca ggagtttgag accagcctga gcaacatggt 93060 gaaaccccat ctctactaaa aatacaaata ttagtccact gtggtggcac acgcctgtaa 93120 tcccagctac tcaggaggct gaggcaggag aatcgctcga acctgggagg cagaggttgc 93180 agtgagccga gatcacgcca ctgcactcca gcccaggcga cagagcaaga ctccatctca 93240 aaaaaaaaaa aaaaaaaaaa agacttttca ataaagatac ttccatgtca tttatgggaa 93300 atctagttct cttgcaacaa tggagaattc ctgattgagc tgagccaaga tgaaggggaa 93360 cacctgcttt tggaacctga gatatgagaa tgaatcatgt atacaattta agaccaaaaa 93420 gagaggccat tgctttcatc cctgctgcca tcctcaattc tcccagtctg gttttctagg 93480 cattttctgt agcccctggt agaactccag gaactccaca gtttgaaaat tcctccctgt 93540 ttccaactcc agtacctctg gttttagtca aagtgctttg cacacatcct ggcctccgcc 93600 tgaccacatc cttcacacag cctgtgggag cctctctgtg agcaaatcaa caaggtttga 93660 cgcatgagag gcaaaggtat gctgactcac tagagaacgg agcagagaga agcaaagcag 93720 aagggatttt ctactgtaat tacccaagat caccagggca gtttgagggt aattgggtaa 93780 ttagtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgacaa ttatctggct cactggctgc 93840 cagatgcttg tcactgcccc tctggccctt gggctgggtt cctggctcta cctgtggact 93900 attaagtgta atgaccctgt cacgcagcag gagcctgtca ttatctccat gttaacatgg 93960 tatttatcca caaaaccctg ccaagtcact ctgtaattga agggcctcca tgtggtcccc 94020 ctgccctggc ttggatggac aaccatgaag gttgtcaggg caggggtggc tttaaaaggc 94080 tgagaaggtg ggctggggtc atcctgggag gctagggagg gatgctgtaa ctaagtcttc 94140 ccccaaccta ttgccaaacc ccaggacccc agaaacagga tccaagaggc attaagacac 94200 ccccaaatca gtcacatttc cacatgaggc taagtcgttg actactgagt aaaatgaaag 94260 ccaactttaa aattttggaa gctcttcgat gataggcgat ctcattacgt aacagggagt 94320 acaactttct agaagtaccc tgtagttctt actctctact tcccagtcac tctgtactat 94380 ctccctcaaa actccaatgc tcacctcttt ccccggggct cccctgcctt tggggctctt 94440 ttctatttgt cacaggcaat tctaaatctt ttctgatgaa aataaaagtt gccctcggcc 94500 tcttccattt ctcttgcttc tcttgccctg tcatctccac aaaaggcacg gacacagaag 94560 tttccttaaa agagttgatg gagtgaagct gtggtcactt atgggtcaat attttgcccc 94620 tggtgcctaa gcagtgctgt tctgagcatc cttatgctta tccttacaca ttcctagact 94680 tggagtgcct gggtatgaat cttttaattt gaaaagatgt tatcaaatag tttctaaagg 94740 gttgtacaca tttaaattcc cactaataat ttatcaggat gcttgttttc ccacattctt 94800 gccaacttgg gttgctaccc cttttaaaaa ttacctttat aattttctga ttatacatgt 94860 gatatatgct cattgcaaaa aaaaaaattg gaaagtacag aaaagtgtaa gagaaaatag 94920 ctgttgtgcc ttcaccccaa gagaagaaac actccccatt tctatagagg ataacagcaa 94980 agaccacgga tggagaccac agtggtcacc tgaggaacct tctggaatcc ctagtgctgg 95040 ttataagatg ttaacgatca cccacttccc cccttcctcc actcctctga tgtctccaaa 95100 ggcttgagat gctttcttca gttggcaggg tctattctga gggagatggt tccaggtatc 95160 tgcacccctg aagtatgtct cccccctcag aacagttctg cagccaggaa gattccacgc 95220 ctgaggattt gtggaatagg attggagcat gaacattcca aaatgtgttt ccagacattg 95280 gccgctggat ccaacagatt ggaaaaccca tctccctggc caagcccaga gccaagtttc 95340 catttctcag ggaccctgac atgccccatt aagcctttgc cctaatgact gctctgcttt 95400 ctgttccaga gacctgaatt ataacaagct gcaggagttc cctgtggcca tccggaccct 95460 gggcagactg caggaactgt aagcgcctct tttggtttct gtgggtgtcc ttctgtccca 95520 agggcagggg ctgaagccag cctgagctgc ctcatgctct tacctcctca cctcccatgg 95580 tccttataaa agatgtagct gtcccccacc ctcccgcccc ggctccccaa gccccaatgc 95640 agctctaagt agtcccaggc ctcattagct tggtttcggg cacttcctgg gggtgtgtca 95700 atagcagaaa tggccctggg gccttccagc ctgggaaaac ccatggcaca agccacctac 95760 tattagcctg ggcctggcac tgcccttgcc ttggttggga tgtgtccaga gaagaatgag 95820 gaaaggaact gagaactggt ttataggatc actcctcagc accttgtccc caggggtact 95880 gagaagttgt ctctggttcc ccaaagccac catttattat tttatttcag cattcgctga 95940 gcacccaatg taccctgcac tgtgtttagt gataaacaca aacaaagggt cagattgaca 96000 tggccctccc caagcatcct tcagcctcat tgaagacaca aagtaacata ggtaaaacag 96060 ggagcaggac aaaatggtat ataactccaa ccataacttg cagagtatta gctaaaacag 96120 cggtgggagt ccagagtaat gtggaaagat cattgtggac aaatcctgag atgagcctta 96180 agggatgacc gggattgagg cgacagtggt ggggaagggg agaggcattt cagaggaaga 96240 aattgtaggt aaagacccag gtaggaagtt gagctggcag agggatgagc acaggtggga 96300 agcagcttta ctggagggaa gctctcagga aacaataaga gctctatcgt acagctagag 96360 tgggaccaga ttaagagtcc tggagactca taaacagagc cccaaatttt caggcagctt 96420 agtatctttt tttttttttt tttgagacgg agtctcactc tgtcgccatg ctggagtgca 96480 gtggcacgat ctcggctcac tgcaacctcc acctcacagg ttcaagcgat tctcctgcct 96540 cagcctcccg agttgctggg actacaggca cgcaccacca tgcccaggta atttttgtat 96600 tcttagtaga gacggggttt caccatgttg gccaggatgg tctctatctc ttgacttcgt 96660 gatcggcctg ccttagcctc ccaaaagtgc tgggattaca ggtgtgagcc actgcgcccg 96720 gccagcttag tatctttata gctcaaacca cccccaccac ctgagaccta tagtactagc 96780 cccatgccac ctcccttgcc tcatccagaa gtttgaaggt gtgaatgcca gatgtaggca 96840 gcccataggg ggcaactggc ccacctagag gaaatagtct ggccgggcgt ggtggttcat 96900 gcctataatc ccagcacttt gggaggccga ggtgggcaga tcacttgagg tcagaagttc 96960 gagaccaacc tggacaacat ggtgaaaccc catctctact aaaaacacaa aaatcagccc 97020 agcgtagtag tgcacacctg tagtcctagc tacttgggaa gctgaggcat gagaattact 97080 tgtacccagg tggcagagat tgcaatgagc ggagattgta ccactgtact ccagcctggg 97140 tgacaaagtg agactctgtc tcaaaaaaaa aaaaaaaaaa aagtctgagt gagaaactat 97200 tggaatttct ccattgccag agtcaccaag gtctaacttt tttcctttga tctggggcat 97260 gagagaagca tgagatttga tctatcaaga gggcatggag ggagcaggat gtgcaaaagc 97320 attgagccgg gaaataataa aacattaggg aagagcgagg agacctaagt tagcagggac 97380 aaaaatatat gggagggggt gtacctgtct atctccatct ctctctctct catctattga 97440 gagaaagctg cttgaggaca gggcccctgt cttagaaact tctaaatccc tcagagcttg 97500 gcccaacatt agacacaaag atggtgttta agaaaaacat aatgagcaga tgagaggaat 97560 aaattaatga acaaacaggc ctgggtgggt ggcactggct ggaggtgtct gagcgtgaag 97620 ctgtgagtga gacagcatga aggtggaagt cacgatgggg atggcaggag gaagacagag 97680 gtgagcagag tccaggggga cagagaagcc agaaacttcc aaagaaaaag agagggaagg 97740 aaaggggcct gctccgcaca ggttatgtac ttttcagtcc aacatgttat ttattttgaa 97800 atgtttttcc caactaacca aacacataat tctggcttcc agactcccag actctcagct 97860 gggtgcctca gtcctcattt tcccatagtg ccatccacag tcattcagcg gcccattccg 97920 caccttatcc ccctggctgc gtgctgttga cacttctgtt tctttatttt agggccttga 97980 ggccaagcca gcctccattt gaggaacatc cctcacttgg gatcccctgg aagctgtgtc 98040 ttttctgtat cttccttttc ctctcacaca cccttggctt cctctgtggg gacccaccag 98100 ggctctcacg acaaatgact aaggagagga ttcccttcct ggggtctgaa ctcagcctct 98160 tgctcttgag ttgctcccac aggcttcctg aggcctgcag ccatgtctcc acccctcact 98220 ctttttacac ttgttaggga aaagagggtg tgtaattgaa caggaagaaa aatcagctgg 98280 gtgcagtggc tcacacctgt aatcccaaca ctttgggagg ctgagacagg aggatcactt 98340 tagcccagga gttctagact agcctgggca acaaagtgag atcttgtctc tacaaaaaaa 98400 tcaaaaaatt agccaggtgc agtggcacat gcctgtagtc ccagctatgt aggaggctga 98460 ggcaggagaa tcgcttgagc ccaggaagtc gaggctgcac tgcggtgagc caagatcgca 98520 tcattgtact ccagcctggg taacagagac cctgtctcag aaaaaaaaaa aaaaaagaaa 98580 gaaagaaaaa tcacaggcag tatttgaatc tgaacctgga atctccaggc caatgcctct 98640 tttgtcttat ggactgtcta gcttgctgtc caaaagccct gtggaattcc atgcctctcc 98700 ctctctatac ctttctccaa atcctcactg gttcctggtg ttgtcttcca gggggttcca 98760 taacaacaac atcaaggcca tcccagaaaa ggccttcatg gggaaccctc tgctacagac 98820 gatgtgagta ctactttctc tggtctctta atgccgaaca gtgtttcagg gaggaggaca 98880 gaggatggga aggcaggagg gacttcccta ggaagcacca gggaggcaga agtatgggag 98940 gggttgcctt tccctgcatg ttctttcctg ggtctacatt gataggagaa gaaagcagag 99000 attggcctta atcccagcag aaaggcctga aaaacccaat taggtgtttg gaccttcttc 99060 ttccagacac ttttatgata acccaatcca gtttgtggga agatcggcat tccagtacct 99120 gcctaaactc cacacactgt aagttggctc ctgaaggctg ctgcagcctg aacttccccc 99180 tttcctcact ccttcttgct ctctcctgcc tatcgcctgc ggatctctcc cttgcaaggc 99240 acaagtccac tgcaaagcgt ggtcttcaag gctgcaggcc tctgctttgc tcagacccgt 99300 tcccacctct ttctaaattt aatagcctcc ctccacctcc ctgaactgtg gcttcttccc 99360 aggtctcaaa gggacaagga aggtccatct ccatagtcaa ggactttcat ctggcctctt 99420 gggttttatc ctgttcacgg tagcttttcc agaaataatt gggagttttg aatatggtgg 99480 gttctcaagg ctggttctcc tgtggccttg gagcccagat ccctgggcac tttgctgtgc 99540 catcgttatc acccttggcc tgaaggtgct ctctatttcc cctctcttat tctctaattt 99600 ccagcccaag tcccacttct tcaaaaagct tccatggggc caggcacaga ggctcactcc 99660 tgtaatccca ccactttggg aggctgaggc gggtggatca cttgaggtca ggagtttgag 99720 accagcctgg ccaacatggt aaaaccccat ctctactaaa aatacaaaaa aagtagccgg 99780 gcgtggtggt gggcacctgt aatcccagct actcaggaga ctgaggcaga ggactcgctt 99840 gaacccggga ggcggaggtt acagtgagcc gagatcgcgc cattgcactc cagcctgggc 99900 aacaagaatg aaactccgtc tcaaaataaa aatacaaaaa ttagccggac gtggtggtgc 99960 atgcctgtag tcccagctaa gctacttggg agactgaggc aggagaattg cttgaacctg 100020 ggaggctgag gttgcagtga gccaagactc agtctcagaa aagaaaaagc ttccatggat 100080 tgctgtcagc ctactgtggc gtcacagtaa catagccgac atctccaaag cacatactta 100140 actttcacgt tcattacggc atttcatctt catcgtaacc ctgtgaggta ggtgttattg 100200 gtccgattta caggtggaac atgaggtact caggccctgt tgggcatgaa cccagaaccc 100260 tttaatttca gattctgggc tcatttctgg accccacgct gcctcctcag ggcttttcac 100320 aacactcaat tttctgcgct gctgttgcat gcaggcccca tgtgggtccg tgtcctcaat 100380 aaaataggag atggtgggaa ggaggaaaac caccagtcta aagcagttta agagaactga 100440 tagttctctt ttttcttttt tcttttttag acaggatctc actctgttgc tcaggctgga 100500 gtgcaatagc gcagtctcag ctcactgcaa cctccgcctc ccgggttcaa gcgattctcc 100560 tgcctcagcc tcccgagtag ctgagattac aggcgcccgc caccacgccc gactgctttt 100620 tgtattttta gtagagatgg ggtttcacca tgttggccag gctggtctcg aactcctgac 100680 ctcaggtgat ccgcccgcct cggcctccca aagtgctggg attacaggtg tgagccacca 100740 cacccggctg agaactgata gttctaataa agttccagca gatgacaatt taggggtgac 100800 cctatctcag gttctaataa gcaaaaccca gcagccaagg gcttctccag aagcacccaa 100860 gctgttgtct attaaccaga ggccattccc atgttgcttg catgtgcggt ttttgcaagc 100920 tgaagcgtga atgctgaggt tacccagctc tgtctgggcg agggtgtgct gagcaaactt 100980 aagcaaatgg ctggttggac cctgctaagt atggctcagc ctccacgagg gcctgcgtgg 101040 ctgacttgag gaagattgca gggaagcatg acattgcccc caaaggagga gtcccggagg 101100 ggagacaaaa tggagaattg agagtcttga tgttgagatg ctctgacccc acccctcaga 101160 gctcattgtc tctatttcca gatctctgaa tggtgccatg gacatccagg agtttccaga 101220 tctcaaaggc accaccagcc tggagatcct gtgagtggct tctctctccc taccttatct 101280 atcgccccag cttcattccc aagagctctt ccactccctg cccctggaag gctgtctagg 101340 ttccttgctt cccttttatc cagcctgtct ctactactgg ctccatcccc ttcctcctga 101400 tgtggcactg cccacaggca aggtcagcag gtggcagatg ggatggcagt gaggaggctg 101460 gtctttctct ctttaccatg cttggagttt tcaggtatta atttagagct cctaacccca 101520 agccacaggt acactgaggg ttgaagtgat ggagaggagg gttgtgaaaa cccgtctttg 101580 cctttctgaa gcttgattcc aacaaagacg ttcttgtatt ttctattatt atctcctgtg 101640 gcatagaggg caggagtgcc gacgcatagt aggccctcaa tacatttaaa tgaatgaaat 101700 gaaacaatcg agcaaacacc cagaatgagt gtcaggaagt cggctgccct gccccacacc 101760 acaaagttca ttcatggcac cactgggact ggggctctcc tctgtgcctc cgttgtctcc 101820 ccgctagatt cactggcatg tggaaaattt tccatgaggg aagtagttat tccccaaaga 101880 tgaccttggc cttgggtcct tcctgggaca ccctgcccga agccaagcct gctctccaac 101940 ctgcccccac agccctctcc agcccagctc ctggttcttc ccttctatcc catctcttct 102000 ccctcctccg aggcacaggg accaaactct gtgcctgtgt cccatggtcc cactacggtg 102060 tttttctgtc tacacttgcc catggacatc ccctcctcta aggtccctac agcagagaag 102120 atttgtgtcc ctgtcctaca ggcccagtgc tgcttgagac cattatgaga catggggtgg 102180 ggatgggatg gggatggctg gagcagagcc ctgggctgtg gccctctctc tccactcccc 102240 tcccccagct cctctaggcc caagggagct tggtgctcag atgccagtgt ctgctcgttt 102300 ttgtccccac agccccaccc cctgccctgt ctctccctcc atttctctct tttgttagct 102360 ccgtcatccc acgacagtat caggtccaga gctggagcgg ggtggctggg aatggcaggg 102420 ccctgggcct ggtgccagct ctgtctctgt tctccctgca ggaccctgac ccgcgcaggc 102480 atccggctgc tcccatcggg gatgtgccaa cagctgccca ggctccgagt cctgtgagtg 102540 ctcacaagaa ttctacagtc ttggcattgt gcccctaccc ccatgtccca caaaaggcct 102600 ctcctgcttc tgtcccactt ggtcatttcc cttcctggag aatggagcag cataggctcc 102660 tgctgagagc ctaccccaga aggaccgggt ttgaggcaca gctctgccac ttactagctg 102720 tattgatttg gactcactct ttaaagtctc tgagcttcga tatcctcgtc tataacatgg 102780 gagtcaattt gtaagggttg gaaaaaatat gtaaagcacc tagtgcataa taccaataaa 102840 tgacataatt actgtaattt tacacaagaa aattagtccc ctcttgggat cttgggactg 102900 tggaggtact taagtggcct ctgaagtcaa ggggagtttt cctctctcct tggactccaa 102960 cttgaatctg aggctcctca tcagggacca tagcccctca gctcaagatg ggtggaccaa 103020 cgcatagtgt gtggaaagag ccctaggctg ggggtccgga gacacaggct ccagactcag 103080 ccttgccact gactggtggt gtgtccttta gtaggtcctg tcccctctgt gggcctcagg 103140 tttctcatct gctgagtgaa agagttgggc cagatgatcc ctaagggcct ccagctctga 103200 tctttcctgc cagctccata aatggtgact gcaggtgctt gggttggcat tgctcagtga 103260 ggtccaatgt gttctctgct ggctggctca gtgcccctct ccctgactct ttgcctgttt 103320 cctcttcacc acgcccctct cacttcctgg gtagtcccat ctaggcccaa tgaggagtgg 103380 gacttagtag gaggaaatgc agagaaagtg aattgaggag actggtgccc tggtgtgaga 103440 tgcctcaaca gccatgtccc tgtggggtcc ccacagcctt cagctaattg acaggaaagc 103500 acagtcctgg ctagagcccc ccgtccccga gcagccctca gatagccacc atttcacccc 103560 agccctggcc tttctctttt ccccagggaa ctgtctcaca atcaaattga ggagctgccc 103620 agcctgcaca ggtgtcagaa attggaggaa atgtgagtct ggggtaggga agaggcaaaa 103680 gcacgccagc cagaccctga gcaagtcctg cagggaggtg tgatgggggg catcagtaag 103740 ggccaatgca cactttgaca tttcttcctt ctcttttctg agtttgttta acccagagtt 103800 tagggctgag agcttggcag aggcctgcac aatggcccta ggactcacag cagaagaccg 103860 acccaggagc tggtgcccaa aaggagtctc aaggtggctc cttataaatc agtagcaaca 103920 cagaaatgca gtagtgatta gaaccagtgg agggacaggg tcacatcagg gcacagtggg 103980 atggaccgga gaaggcttta aggagttgga agcaccctgc attttggagc atgttctctc 104040 cttttccata cctctaaaca cgcacatgca tatgtgtgta tgtgtttgca tattacacac 104100 agacatacat aatcatatgt ggaagaacaa gcctgggatt caaaggtcac aaagaaaata 104160 tcacttgaaa aattctctaa gctgtattaa cccagctcct gttttctcaa gccagcatgg 104220 cctcttgagc ttccctgtgt ctgtaccctg cttctaggca ggaccatgca ttttggggct 104280 ggggcagggt acagatatga gggtctcctg tttgtctagg tgccaaggga aggagggtct 104340 tctgcatggc tgaggcccag ggtccatctc ccattcaacc agtctactga gctctgcttc 104400 ccagttccgg ccagtcactt gggttgggcg gtgctctgct tctgggctag ggtctggagg 104460 acccatggtg atgggaggtg tgggggccag tttggctggg ctgggagcca ggcccagtgg 104520 gaacactgga agctgactcc cttttgggcc acagggatgg ttttgtggct ttgtctcctg 104580 acccacttac ccaaggcaac cggatcccct tgtgctccct ttaattctgg tgacttcctc 104640 gggctgggcc ctttcttcct gtgccaggag aagtgggggg ctctactttc ttcctcccag 104700 tgctcggggg gcatggagcg gagccagggt ctgagcctgc cggctcatcc agcctctctt 104760 gctgccctag cggcctccaa cacaaccgca tctgggaaat tggagctgac accttcagcc 104820 agctgagctc cctgcaagcc ctgtgagtac ccacatccag gggtgggcca tggaggagcc 104880 accgtgtccc tctgctaggc atgctcattg tcatagcaca tgtgtacaat ttaagcaaat 104940 gtgacatgca catcgcctcc cagctccaca gcccccaccc ccagcacaca cacacacttc 105000 tctagctggc acccaggagg tcttgaggct ctggagccaa gctgtcatgt ccctgttcct 105060 ggggccacag ccagctcagg agctgggcag cagcaggtac cttccccaaa aggcaaaggt 105120 tactgttact gggggcaggt cagatggggg tatgtgaggg ggagcccatg atgggaacag 105180 tgagtcctcc acatgttact gtcccctcct gtcacaccct ctctgcctgc agggatctta 105240 gctggaacgc catccggtcc atccaccccg aggccttctc caccctgcac tccctggtca 105300 agctgtaagt gcctgctgca ttctcctcca ggatgctggg ggccagtcgg gactatgggc 105360 tggccaatgg aggcagaagg gccaccccag agcaggaaca tgcatatccc agggagatgg 105420 gcctcagcct aatcaatcat tctgacaatc cgtttgacag agtgtggggc ctgataaaca 105480 gagatcctgc ctcttgtaag ctctggaaca tcatctgcta atgttctgca gtattttggc 105540 agcctcagcc ctcctcatcc cccctgcctc ggtcccatcc ccatcctctc tatccaccaa 105600 caaagaggat atattgggcc tggaatccct tcacacatag gagggtggaa gtagatgtgg 105660 gagccagggt ctcagagagc tcaggggttc cctgcttgag cattgctgta gaggactgcc 105720 attctgtgag gcagcagggg acctcaggtc ctttaaatta cataggatgt aaacagagag 105780 tgagcagtgt tggctacatg gtcattatgt cagggctgga aggggcctct gctgcagctg 105840 ccatgtactg tggggcaggt tattcactgc gcaagcactc ctgcccaaga gggagctgac 105900 atcctgccac ctccttgcca agctggactc ctaaaagcac atctacctag actgggccct 105960 tttctggtta acacaaagac ttcttttggg ctagctccag ccctggcctc agagaccatc 106020 cagccccacc cgcttcactt cacaggtgag gcctccaagg cccaggagga gaagtttact 106080 caaagccaag ggccaggact cccagcaggc acacacattc ctcctcaggc tggggccgtg 106140 gctttgcaaa actctcacct ccgtcttccc tctcctgttt gctctagtcc tgccagatag 106200 gtagggtgga tatttttatc cctttgaaag atgaggacac tgaagctcag gcagatttgt 106260 ggctcccctt ggttacacag cgagttggtg acagtgccag gtttcctgcc ttccagtcca 106320 gggctgtttc cacgacgcac agtctgtggt cttagcgttt ggtttggaac cctgggcttc 106380 ctctaatgcg cctccagtag aagaaaatgc atgtcctcgc ctctggctac aaagcagtcc 106440 ctacaagatg gaatggatga cccaatccct gcttgtagcc agatgaccag ctctgtccag 106500 tcccagactc agccctctta tatcatcaga aaagtaggag gaaaggcagg gggatgggga 106560 taaacgacaa caggaaggtt ccccaagtgg gtgggcaggg cttcctggat ggcacttaga 106620 actgggcaca ggaactgtgg ttgagggagc tctgttacct acaaattggg acagcttggc 106680 attaagttag aacagtccct ggtatatagt tggcaccgtt atgtattgaa tgaatgagca 106740 aataaaagaa tgaacaacct gcagatggtg cctctagcaa ctcttggctg ccctcctccc 106800 tcggcattca atgctctctt ctaagcttct ctcctgtcct ttgctcctct cctcttgctt 106860 ccatcccagg cacactggcc acattctcag cacagccctg ccccctcagc aatcccactg 106920 actgccaata accctgacca tccactgtcc tagggacctg acagacaacc agctgaccac 106980 actgcccctg gctggacttg ggggcttgat gcatctgaag ctcaaaggga accttgctct 107040 ctcccaggcc ttctccaagg acagtttccc aaaactgagg tgagggactg gctttcccca 107100 acacctgggt aggccagctg gagaccctgg agggacagag agaaagccag atggccccac 107160 cctgagaaga gcctagaggc ttagggtttg accaagattt agtggaaggg ctcacctgac 107220 cacctacaga ccaagagggg cctggttcat gcctcccctt cctccttatc tccttggtgg 107280 cattttcccc tgtcccaagc tccaacgcta gtgagcactg ggacctgaga gtgatagaag 107340 taagatgatg tgaagacccc ctagccctcc ttgcacccca tctggttcca catgtccaga 107400 ttatctgtcc tcacttgggc agcatctgag cccctggaat cagggcagag cccaggacac 107460 tgatgggtgg aaaacaagct gcgcagccag gccgtggtgc taatgacatc caggcctctt 107520 ccatgcagca gcagtggtgc agcctgcatg gcccaagcct tgtctgctgc acactaatga 107580 gatgccctta gggcctctgc acttaccctc agagggcacc aaatccagct ccaatatgcc 107640 cagaggtggg ggagggatag ggtgcaggtc tcactgaggg ccagagtgaa gaatcaccag 107700 aagaatccat gtggccttaa atataaaaga ggaggtgttg cctgggagaa gaagcagagc 107760 actggcagtg ctgtgtgagc ttggggaagc ctctgcccgc tctgagccag cagagaggac 107820 tccttaaaca atgggcagaa tgtttctttc cccatctcac ttgccaagat gaacaaagaa 107880 agacagaaat gcctccagag cccttcagat caggagtcag gctgcaaggc ccttacaaag 107940 aatgcctagg agactgccag cacagtgaac agggaacttg ggagttgaag gacagacact 108000 gagtgcccag cccctgggtt gcagatctct taaatcagac ttaggtctta gaccccaaag 108060 atgctgaggc agccaatcag cctgcctctc ccccaccagg atcctggagg tgccttatgc 108120 ctaccagtgc tgtccctatg ggatgtgtgc cagcttcttc aaggcctctg ggcagtggga 108180 ggctgaagac cttcaccttg atgatgagga gtcttcaaaa aggcccctgg gcctccttgc 108240 cagacaagca gagaaccact gtgagtgacc aggggccctg ggttggggag ggtagtgggc 108300 tgtggagagg aagttagagg ggatgcttgg gggacctgag agggaatctg acagcagctg 108360 cagagggaag ccctacagag gtgggaggag ctgcctgtga cctgggagga aactctacct 108420 ctgtttgcac tttgtctctc caagactaca cccagaggtg cttgggacaa agtaatgatg 108480 gtagcaatgg ctgtggcaat gtctcatctg cctagccctt tgcagttcac aaagtgcctc 108540 tcacacacca tctcacttaa ccctcccctt gtggtccaca tgtttcttct ccttcaacct 108600 cagggtattc tcagagaaga cagacccctt attttatcaa tctgagccaa taggatttaa 108660 ttaaatcaat tgaattaaac ctacttccag aaaaaaagaa tcccctcata atatgtccta 108720 gaaccaaaag gaacagtgta gggttattgc tgtccaagtc catcctgcac ctccagtctg 108780 ccccatgagt ctgaaagtga gacttccgtg gccgccaggt gcaagagacc ttcactgaga 108840 agggccattc cagtgcaggc tgtcttgggc catctgtgtt tctggaagag ttagggtaaa 108900 aggcacatgt tggaggcagg cagccaaggg ggttctgagg gatcatttac taagtggtat 108960 cttagatacc aagggcagat aaaagggctt ccctctcccc ctgcccgtat tacagaacac 109020 aagcagaatt aggagggaga gagtaggggt cctctgcttt ggggtattct ttttccatct 109080 ctaactctcc acaatatttc aatggagaaa ataaacaaac gaaaaatcaa atataaacat 109140 gtaagaaaaa catctcatta cagcgtagac aaaactgagg tccaatccca actacaacat 109200 tcaccagttg gattgcattg gaccagtcat gtaaccatac tgagcctcac tttccttatc 109260 tttaaatgag aaagataatt ggctgggcgc ggtggctcaa gcctataatt ccagcacttt 109320 gggaggccaa ggcgggtgga tcactttagg tcaggagttc gagaccagcc tggccaacat 109380 ggtgaaaccc cttctctacc aaaaatacaa aaattagcca gtcatggtgg catgcgcctg 109440 tagtcccagc tacttgggag gctgagacag gagaatggct taagcccagg aggcggaggt 109500 tatagtgagc tgagatcgca ccactgcaaa ctccagcctg ggtgacagag tgagattctg 109560 tctcaaaaaa ctaaataaat aaataagtaa atgagaaaga taatggatag ctcataggct 109620 gttggggaag actaaaagat tccataaatg atgaagtatg ctcatcgcta ctactgagga 109680 ctggtgcctt ctggtgtaca tgtataggga gagtgcaact gtggagtccg gggcacagtg 109740 tatggagggc aaatggtggg caggcaacct gggggtctct gagagccttc ttcccaatgt 109800 attaacacac ctccctaggg agggtatgag gacccgatga ggagcagccc agcagcctgg 109860 ctggctctgg gccagcagac agctgtcccc gggggacatt cccggatgtc tgctgtacac 109920 tccctcaggg ggctggaagg aagaatggaa actttccttc ctcaaggtga aactttccac 109980 cgagcttagg tcttggagaa ccacagggcg tcaagatcgt gggctgcctg ccgcctgtaa 110040 cgggagctag ggcagggccc cccacttcct gtgggagcta atgagcctcg agaaggaaag 110100 ccaggagctg ctgctccagc cagattcgat ttcctcctcg tttgaagagt ggttacttaa 110160 caccttggct gtgattttcc cagcctcaga cagagtccac gggtttcacc ccttgtgttt 110220 cattcatgca ttttgatttc aaagaattgc tgaacttctt tctggagagc aggggtgggc 110280 agggagtgaa aggtagagag gaggaaatgc caggactctg tgggcacgac tcagcctccg 110340 acctgccctg atggtgtctg ggccaggagc agcttcacag tcttcctgcc tcctgggaaa 110400 gctcccaact gatggtgttt accctttaag tgaggaggaa ggatccacac aggaaacaga 110460 ctttgtgaac caaagatgga acaagtgcaa caaacagagt cacctatcca ttgccctgta 110520 tgatggagga agagcccaca ggatgctgtc tcttaaacct gaagattgta cagttacctt 110580 ttgaagacaa gccattcttt ccccaacttg ggtcttcaga cacatttcca gtctgacctg 110640 agaaacttgt cttattcttt aaaaagcctt tcagtgatgg attatcactg caaaaaatgt 110700 tttgtcttta tagattataa aatgttctag attctcattt caaaattcgt atagaatttg 110760 tggacctcca gcatgtctgc aaccccctag agttcacagg ccctagtttg agaaacactg 110820 caaaagtgaa taagaaaaaa aaagtgtaag gccgggcacg gtggctcacg cctgtaatcc 110880 cagcactttg ggaggccgag gcaggcggat cacaaggtca ggagatccag accatcctgg 110940 ctaacaccat gaaaccccat ctctaaaaaa atacaaaaaa ttagccaggc gtggtggcgg 111000 gcgcctgtag tcccagctgc tcaggaggct gaggcaggag aatggcgtga acccaggagg 111060 cagagcttgc agtgagccga gatcatgcca ctgcactcca gcctgggcaa cagagcgaga 111120 ctccatctca aaaaaaaaaa aaaagtgtaa agcctggact tagaattgaa aagaacatta 111180 aagatgatct agtccagcct ctgactcagg tagaagtgct ttctctccca ttggggactt 111240 tctgtttagt atcatcagtg acaaattatg ttcagctcat gcatctgtaa atctcctgaa 111300 ccctccccac cccctgaccc cccaaagtaa tcagtccctt gcctgggaaa gcatgatggt 111360 taaacatctg ggctctgtag tcagacaaac ctgcatctga attctagtct attgctgtga 111420 ggctgcaggc aagccactta acttcaatga gcctcagtct ccttatctgt cacctgcctc 111480 acatggatgt tgtgaaggtt aatgggacca tgaagatgag gactgactgg gcacagtacc 111540 tggtgccgaa taagaactca agaaatgtta acccttattg ttcagtgaat ctattagctg 111600 aaaatttttt tctaattgat acataataaa tgtatgtatg gggggcacat gtgattattt 111660 aatacattca tataatgtat ataaagatca aatcagagta atcgggatat cctttgcctt 111720 aaaaatttgt cttttcttta tgctaggaac attcaaatta ttcccttcta gctagtaaat 111780 aaatctgtta gttgatggca ctcattgtag catgacaatt tgtttcctta ttagtctcca 111840 caagctcttt gagagcaggt ccatctctta ttcatccctg aaattcttct gcatttgccc 111900 agtgccagaa ttgcactcag cagatgtgga cctactctgc acctgttgaa ggtggggtgt 111960 tcctttgctg gacagctcta attgtgggaa cttactcttg ttaggaagta gtagagtgtc 112020 tccattcgaa tcataatttg cctcgttcta cagtccatcc attgatcctg gttctaccct 112080 ctggggcaaa acagcatgta gaagtcaaat tcctttaccc ctaacagtcc ttcagccctt 112140 tgaggacggc catgataccc ccacttactc ttacctcctc caagttaaac accgcccagt 112200 tgctttgact gtttctctca caaatggctt ctgctctccc accccctagt tcactcgttc 112260 acgtctaaac acactgcagc tcactgatgt ttttcttaat gtgtagtttt cacaactgga 112320 cacaaaatta cagtgacagc ccagccagag tagagaatgt gtgatcacta gcatacatta 112380 ttgaaacagc ttccactcca actcgaagca gcacttgtgg gtaaggctga ctggagagga 112440 catagagccc tgactaatag aaagagaagg tctaggtgaa atgtttgctg tgcactcttc 112500 agtgcagata atccacatgt ggataatagg aagctgacca aatctttacc cccaagggta 112560 gaaatggatg tgccgggttg ctagaatgaa cagtgctgag cagagttagt aagggacatc 112620 ttaaaggagg gggcatttgg agaaaggtag ggcttgactg acaccaagac ccaggaccct 112680 gcatcacttt gtctctcctt tcagatgacc aggacctgga tgagctccag ctggagatgg 112740 aggactcaaa gccacacccc agtgtccagt gtagccctac tccaggtgag gtggtgtctg 112800 gggaatgggg acgaggggga atggagaaag ggcacccaac cacctagttg aggtattagt 112860 tcagcctggc atgtctgcag ccacattctt tgcctgactc ccagcctgct gcaagagggc 112920 cctcctttcg taattcctgg ggaaggaact ggagttcttt cccttgggtg gctgcttgag 112980 gggagtagca tgaatggagg cgaagtcaag caatcagcca attagcattt atgcatccag 113040 gataaaacta ctgttacaca ggagagaagc aggaaagaat gccttgaccc cattgctcag 113100 ggtggcacgt gggactgctt ttgagtaatc cctcagtcta agacccatac cacagaatcc 113160 tagagccagg tgggactcca acaggtcacc ttctccatcc ctccctgctc catcccagga 113220 agattacagt ctctgttaaa actctgcaca gtggaaaaac tccatagtat ccctgggtaa 113280 gtccttacag caccatctaa cgcttatagt tgcggaagcc tcctttagca tctaacctaa 113340 atccatcctg ctataaagaa agcaaaaaag aaaattctct ttctctttgg tgcaattcaa 113400 cttgagacat ttagagtgag gaagtccctt cgcttgcata taaaatgcta acaggccaca 113460 gaaggtggca agcagcatgc tacttaggca aggatgggat tcttcagaat gaacagaagt 113520 gatgaacagt ttgcttcttc tatttcgttc cacctccgtt ttccagtttt ccagctttct 113580 ctggcccagg acccttgcct tctcccttta ccctctctaa agcctataga gtgattgcct 113640 ggccttgccc agcccgagtt ttcagtgctt ggtgacaaca gcacgccctc tagtgccagc 113700 ttggggccag agcactaacc caacctggag gaagcaggtg ctgcttctga cttggaaaaa 113760 cacacatgct gcattcacaa tgccatggaa atttcacctc tgaaaaccag taatcagcag 113820 ggatgaggta aaagaggaag ttccaaaaga caaagtccaa ctcgagcttg cttctaaaat 113880 aactcacacc aaatggtcaa ataattatgg tacattttat ggtacataaa atatgttttg 113940 agtaatataa aaatatttct ctgggaaaaa atttgggaaa gtctgcttga taaaaatgtg 114000 acttaaagag aacgagaaac gcatatacag ttgtcttagt ccgcttcgtg ttgctataac 114060 agaataccta aggctaggta atttataaag gaaagagttt tgtttgactc acaattctgg 114120 tggctgggag gttcaagatt ggacagctgc atgtgatgag ggcctcaggc tgcttcaact 114180 tgtgatggaa agcagaaggg gagtgggtat gtgcaaatag atcacagggt gaaagagaag 114240 ccagactccc tttaacaacc cactcctagg gaactaatcc attccctgga ttgattgaga 114300 actcactcac cctcacagaa ggacattaat ctgttcctga gggatctgtc cccatgaccc 114360 agacacctcc cactaggccc cacctccaac attcccacat tgggggattc catttcagca 114420 tgagttttgg tgcagacaaa ccatatccaa atgataacaa cagtaaaatc ttaatttagc 114480 ttgaaaacct atatgtatgt atgtattttt taaagactgg acggaagcat accaaaataa 114540 tgagttatct ctgagaggtg aagtctcatg tcattttaat gttttcatgt tatattttca 114600 aattttctac agttatcacc tattatattt ggtaataaga aaaaaaaggt ttaaaaatag 114660 ctcccacaaa agcctctcaa attcccaagc taccaagaaa cacattgaag aacaagagtg 114720 gaaagccaaa gaaaatgaag aaataatatt tccaaagtta caaggtgatt taggaacaga 114780 ctttggagag agggatggaa aacaccagga tctcacgcat ctcgtgtgga cctaggttgg 114840 aaaaccccag aagtcatctg tattttgatt tactgtactt gaggcaattt acctccgaaa 114900 tactgattga ctgacaagta ttcaatgtgg tctgtacccc tctcacagag caagggagag 114960 ccagcctggt ataatatttg gtcaggactc tcccacagtg gaggacttag tggatcactg 115020 gggactgact gagagaaaca gagcctcagg catagactgg ttactgtctg ggcagtccct 115080 catgcctctg tccctaccca ccatagctcc caattctgat accctctcca ctccttgatg 115140 gcagcctgtc cttcctttct ttccagtgct gtccttcact taacatttcc tcaatatctg 115200 ggtttgtact gtattaattt gtaccatgtg tgtgtttttt tgtttttttt tttgtttttt 115260 tggggagaga gagagtcttg ctctgttgcc caggctggag tgcagtagag caatcttagc 115320 tcactgcaac ctccgtcccc cgggttcaag tgattcttct gcctcagcct cccaagtagt 115380 tgggactaca tgtgcgtgcc accacaccca gctaattttt tgtatttgta gtagagatgg 115440 ggttttgctt tgttggccag gctggtctca aactcctgac ctcagacaat ccatccgcct 115500 cagcctccgg aagtgctgga attatagtcg tgagccacca tgcctggcct atatgcgttc 115560 tatgttctgg tgtattgaga ggagggttag ggttcacccc acttgcagct cttcggaaga 115620 agagtcgtgt atgtcccaca gctgggagtt ctctaaggtg agagaacctc caacttggag 115680 ttctctgggg ccagaaacag ggtcagcctc ccttagactg ggagttttcc gagggcaagg 115740 gccatgttca tctccttttg attggaaatt ctctgaatac agaggctgac cttggtcctg 115800 aagacctggt cccaccatcc tctggcccag ggttaatgtc tgatctctcc tacaggcccc 115860 ttcaagccct gtgagtacct ctttgaaagc tggggcatcc gcctggccgt gtgggccatc 115920 gtgttgctct ccgtgctctg caatggactg gtgctgctga ccgtgttcgc tggcgggcct 115980 gcccccctgc ccccggtcaa gtttgtggta ggtgcgattg caggcgccaa caccttgact 116040 ggcatttcct gtggccttct agcctcagtc aatgccctga cctttggtca gttctctgag 116100 tacggagccc gctgggagac ggggctaggc tgccgggcca ctggcttcct ggcagtactt 116160 gggtcggagg catcggtgct gctgctcact ctggccgcaa tgcagtgcag cgtctccgtc 116220 tcctgtgtcc gggcctatgg gaagtccccc tccctgggca gcgttcgagc aggggtccta 116280 ggctgcctgg cactggcagg gctggccgcc gcactgcccc tggcctcagt gggagaatac 116340 ggggcctccc cactctgcct gccctacgcg ccacctgagg gtcagccagc agccctgggc 116400 ttcaccgtgg ccctggtgat gatgaactcc ttctgtttcc tggtcgtggc cggtgcctac 116460 atcaaactgt actgtgacct gccgcggggc gactttgagg ccgtgtggga ctgcgccatg 116520 gtgaggcacg tggcctggct catcttcgca gacgggctcc tctactgtcc cgtggccttc 116580 ctcagctttg cctccatgct gggcctcttc cctgtcacgc ccgaggccgt caagtctgtc 116640 ctgctggtgg tgctgcccct gcctgcctgc ctcaacccac tgctgtacct gctcttcaac 116700 ccccacttcc gggatgacct tcggcggctt cggccccgcg caggggactc agggccccta 116760 gcctatgctg cggccgggga gctggagaag agctcctgtg attctaccca ggccctggta 116820 gccttctctg atgtggatct cattctggaa gcttctgaag ctgggcggcc ccctgggctg 116880 gagacctatg gcttcccctc agtgaccctc atctcctgtc agcagccagg ggcccccagg 116940 ctggagggca gccattgtgt agagccagag gggaaccact ttgggaaccc ccaaccctcc 117000 atggatggag aactgctgct gagggcagag ggatctacgc cagcaggtgg aggcttgtca 117060 gggggtggcg gctttcagcc ctctggcttg gcctttgctt cacacgtgta aatatccctc 117120 cccattcttc tcttcccctc tcttcccttt cctctctccc cctcggtgaa tgatggctgc 117180 ttctaaaaca aatacaacca aaactcagca gtgtgatcta tagcaggatg gcccagtccc 117240 tggctccact gatcacctct ctcctgtgac catcaccaac gggtgcctct tggcctggct 117300 ttcccttggc cttcctcagc ttcaccttga tactgggcct cttccttgtc atgtctgaag 117360 ctgtggacca gagacctgga cttttgtctg cttaagggaa atgagggaag taaagacagt 117420 gaaggggtgg agggttgatc agggcacagt ggacagggag acctcacaga gaaaggcctg 117480 gaaggtgatt tcccgtgtga ctcatggata ggatacaaaa tgtgttccat gtaccattaa 117540 tcttgacata tgccatgcat aaagacttcc tattaaaata agctttggaa gagattacac 117600 atgatgtctt tttcttagag attcacagtg catgttagtg taataaagag ataagtccta 117660 cagtagtaaa atctattgaa ctttgtttac ccagttccct gaattatttg gtaacaggtt 117720 tttgtctgct tatttggttt tcctggaata tctattaaca tgacagaact tagcactgtt 117780 ctctcccagg gagctcatct ctgtaccacc tagtgctacc gagtgacatc acaaaatgcc 117840 accacttcct gaaagccccg atcctagaat gaactgccag aggtaattga tgactccatc 117900 caccagtaga gacactcagc actttacctc ccacctctct tatcctatgc tgggaaggat 117960 gaggagaagc ctgccgttgt ggttccagac tcagacccct gagttggggc agggagaaag 118020 ggatccctgt gtgttctctc accaccctgc catcaacccc agcaagccca agcaccagtg 118080 aaagtttgtc tgctgcaaga gggatagtag ggttcctggg tcctggcagg gctgtctgga 118140 ttccctctgg ctctttgttg ccttcttatg cttctccacc agatgcccct gccacctcct 118200 gtcacaagcc aggtcctgcc tactctttgg gacaaggagg ccttagccag aaaacaggag 118260 agatttgaat tgtagatatt gcattttagg aaattgtagt tttctgggac cctgggtgtc 118320 tcagataggc cgctgaccct tctgcctctc ccctcatgac ctttctacct gtggtttacc 118380 acgtgtctct gagcatttcc agaatgcccc cctcacccag ccccaaacac acacatattc 118440 aatctgagag tgaggcggta agagctgggg catccttcca gacattaaag gtaattcttg 118500 gagacataaa aagagaatcc aagactcagc aaagggcaag acttgctcta gagtacccca 118560 acatcagtca catctacaag acaggagtct agaattctga gaccagatgg aaaacaatct 118620 tcttcctgaa ttctggcagg tcctcagcca ggccctatcc ccaggggctc cactattaaa 118680 acaacaaacc tgaggaaaac ccttcttcta attttgtagt tcaagtttct gcagcttagc 118740 ttctctgcta tctccctgct gtccccaatg cctagtggaa attctgacac aggacaaaaa 118800 ggctgcctag gctgaggtag gtagggtagc tccatgggtg aaacttgcta catttctaaa 118860 tcttatctga ctaattgcct ggtctcttat aggctctgaa gcagaatgcg ctgaccttgt 118920 aatcccagaa gaatcttagg gtcctaccat gaggccagcc tcgctgtagg tactcgctaa 118980 tgatggcagg tgtatttatc agttgtttat ttgagaaccc tgctctccca atttccccag 119040 ggctgatcaa gagcccttaa aggaaggagt ttgctctact ttggatactg agaattggtg 119100 actcttatca gggaaaggag ttctttgctt cagggatgac aaaacttgta caatttttgg 119160 gagccttgac ttgtaaaaag tcaccccaga tcccttctca cttcttgccc acgccatgtg 119220 atcattgttc cccttggagt tagggagtct gtgttgaggt ggagcacagt gaccatgtgt 119280 tagaggttta ggagttacag gaactgctcc ctacaggtgg cctcttctat gaaaagggga 119340 ccgatgattt ttggctgcaa gctgaagggt agcaccactg tcaccttatc acctgcttct 119400 aaagatccag acgtctgctg gccagaggag catctgctga accttctcat tagaggaaag 119460 gtgtggagga agggagtgga ggtgtttgcc atgaactatt ttcattattc agaaagtctg 119520 gtcgatgtac acttacccga aataaagctg agcagtacag tttctttgtt ttttgatttt 119580 gactgaattt ctagca 119596 4 3349 DNA Human 4 cgcttgtgtc taactgatgc tcctaatgcg gaagcccctg aaaggcggtt gtggtgcaaa 60 ggaaaaccca caggccaagg aatgggaaga ccaaggttga cacttgtttg tcaagtgtca 120 ataatcatct ctgcccggga cctcagcatg aacaacctca cagagcttca gcctggcctc 180 ttccaccacc tgcgcttctt ggaggagctg cgtctctctg ggaaccatct ctcacacatc 240 ccaggacaag cattctctgg tctctacagc ctgaaaatcc tgatgctgca gaacaatcag 300 ctgggaggaa tccccgcaga ggcgctgtgg gagctgccga gcctgcagtc gctgcgccta 360 gatgccaacc tcatctccct ggtcccggag aggagctttg aggggctgtc ctccctccgc 420 cacctctggc tggacgacaa tgcactcacg gagatccctg tcagggccct caacaacctc 480 cctgccctgc aggccatgac cctggccctc aaccgcatca gccacatccc cgactacgcg 540 ttccagaatc tcaccagcct tgtggtgctg catttgcata acaaccgcat ccagcatctg 600 gggacccaca gcttcgaggg gctgcacaat ctggagacac tagacctgaa ttataacaag 660 ctgcaggagt tccctgtggc catccggacc ctgggcagac tgcaggaact ggggttccat 720 aacaacaaca tcaaggccat cccagaaaag gccttcatgg ggaaccctct gctacagacg 780 atacactttt atgataaccc aatccagttt gtgggaagat cggcattcca gtacctgcct 840 aaactccaca cactatctct gaatggtgcc atggacatcc aggagtttcc agatctcaaa 900 ggcaccacca gcctggagat cctgaccctg acccgcgcag gcatccggct gctcccatcg 960 gggatgtgcc aacagctgcc caggctccga gtcctggaac tgtctcacaa tcaaattgag 1020 gagctgccca gcctgcacag gtgtcagaaa ttggaggaaa tcggcctcca acacaaccgc 1080 atctgggaaa ttggagctga caccttcagc cagctgagct ccctgcaagc cctggatctt 1140 agctggaacg ccatccggtc catccacccc gaggccttct ccaccctgca ctccctggtc 1200 aagctggacc tgacagacaa ccagctgacc acactgcccc tggctggact tgggggcttg 1260 atgcatctga agctcaaagg gaaccttgct ctctcccagg ccttctccaa ggacagtttc 1320 ccaaaactga ggatcctgga ggtgccttat gcctaccagt gctgtcccta tgggatgtgt 1380 gccagcttct tcaaggcctc tgggcagtgg gaggctgaag accttcacct tgatgatgag 1440 gagtcttcaa aaaggcccct gggcctcctt gccagacaag cagagaacca ctatgaccag 1500 gacctggatg agctccagct ggagatggag gactcaaagc cacaccccag tgtccagtgt 1560 agccctactc caggcccctt caagccctgt gagtacctct ttgaaagctg gggcatccgc 1620 ctggccgtgt gggccatcgt gttgctctcc gtgctctgca atggactggt gctgctgacc 1680 gtgttcgctg gcgggcctgt ccccctgccc ccggtcaagt ttgtggtagg tgcgattgca 1740 ggcgccaaca ccttgactgg catttcctgt ggccttctag cctcagtcga tgccctgacc 1800 tttggtcagt tctctgagta cggagcccgc tgggagacgg ggctaggctg ccgggccact 1860 ggcttcctgg cagtacttgg gtcggaggca tcggtgctgc tgctcactct ggccgcagtg 1920 cagtgcagcg tctccgtctc ctgtgtccgg gcctatggga agtccccctc cctgggcagc 1980 gttcgagcag gggtcctagg ctgcctggca ctggcagggc tggccgccgc gctgcccctg 2040 gcctcagtgg gagaatacgg ggcctcccca ctctgcctgc cctacgcgcc acctgagggt 2100 cagccagcag ccctgggctt caccgtggcc ctggtgatga tgaactcctt ctgtttcctg 2160 gtcgtggccg gtgcctacat caaactgtac tgtgacctgc cgcggggcga ctttgaggcc 2220 gtgtgggact gcgccatggt gaggcacgtg gcctggctca tcttcgcaga cgggctcctc 2280 tactgtcccg tggccttcct cagctttgcc tccatgctgg gcctcttccc tgtcacgccc 2340 gaggccgtca agtctgtcct gctggtggtg ctgcccgtgc ctgcctgcct caacccactg 2400 ctgtacctgc tcttcaaccc ccacttccgg gatgaccttc ggcggcttcg gccccgcgca 2460 ggggactcag ggcccctagc ctatgctgcg gccggggagc tggagaagag ctcctgtgat 2520 tctacccagg ccctggtagc cttctctgat gtggatctca ttctggaagc ttctgaagct 2580 gggcggcccc ctgggctgga gacctatggc ttcccctcag tgaccctcat ctcctgtcag 2640 cagccagggg cccccaggct ggagggcagc cattgtgtag agccagaggg gaaccacttt 2700 gggaaccccc aaccctccat ggatggagaa ctgctgctga gggcagaggg atctacgcca 2760 gcaggtggag gcttgtcagg gggtggcggc tttcagccct ctggcttggc ctttgcttca 2820 cacgtgtaaa tatccctccc cattcttctc ttcccctctc ttccctttcc tctctccccc 2880 tcggtgaatg atggctgctt ctaaaacaaa tacaaccaaa actcagcagt gtgatctata 2940 gcaggatggc ccagtccctg gctccactga tcacctctct cctgtgacca tcaccaacgg 3000 gtgcctcttg gcctggcttt cccttggcct tcctcagctt caccttgata ctgggcctct 3060 tccttgtcat gtctgaagct gtggaccaga gacctggact tttgtctgct taagggaaat 3120 gagggaagta aagacagtga aggggtggag ggttgatcag ggcacagtgg acagggagac 3180 ctcacagaga aaggcctgga aggtgatttc ccgtgtgact catggatagg atacaaaatg 3240 tgttccatgt accattaatc ttgacatatg ccatgcataa agacttccta ttaaaataag 3300 ctttggaaga gattaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 3349 5 915 PRT Human 5 Met Gly Arg Pro Arg Leu Thr Leu Val Cys Gln Val Ser Ile Ile Ile 1 5 10 15 Ser Ala Arg Asp Leu Ser Met Asn Asn Leu Thr Glu Leu Gln Pro Gly 20 25 30 Leu Phe His His Leu Arg Phe Leu Glu Glu Leu Arg Leu Ser Gly Asn 35 40 45 His Leu Ser His Ile Pro Gly Gln Ala Phe Ser Gly Leu Tyr Ser Leu 50 55 60 Lys Ile Leu Met Leu Gln Asn Asn Gln Leu Gly Gly Ile Pro Ala Glu 65 70 75 80 Ala Leu Trp Glu Leu Pro Ser Leu Gln Ser Leu Arg Leu Asp Ala Asn 85 90 95 Leu Ile Ser Leu Val Pro Glu Arg Ser Phe Glu Gly Leu Ser Ser Leu 100 105 110 Arg His Leu Trp Leu Asp Asp Asn Ala Leu Thr Glu Ile Pro Val Arg 115 120 125 Ala Leu Asn Asn Leu Pro Ala Leu Gln Ala Met Thr Leu Ala Leu Asn 130 135 140 Arg Ile Ser His Ile Pro Asp Tyr Ala Phe Gln Asn Leu Thr Ser Leu 145 150 155 160 Val Val Leu His Leu His Asn Asn Arg Ile Gln His Leu Gly Thr His 165 170 175 Ser Phe Glu Gly Leu His Asn Leu Glu Thr Leu Asp Leu Asn Tyr Asn 180 185 190 Lys Leu Gln Glu Phe Pro Val Ala Ile Arg Thr Leu Gly Arg Leu Gln 195 200 205 Glu Leu Gly Phe His Asn Asn Asn Ile Lys Ala Ile Pro Glu Lys Ala 210 215 220 Phe Met Gly Asn Pro Leu Leu Gln Thr Ile His Phe Tyr Asp Asn Pro 225 230 235 240 Ile Gln Phe Val Gly Arg Ser Ala Phe Gln Tyr Leu Pro Lys Leu His 245 250 255 Thr Leu Ser Leu Asn Gly Ala Met Asp Ile Gln Glu Phe Pro Asp Leu 260 265 270 Lys Gly Thr Thr Ser Leu Glu Ile Leu Thr Leu Thr Arg Ala Gly Ile 275 280 285 Arg Leu Leu Pro Ser Gly Met Cys Gln Gln Leu Pro Arg Leu Arg Val 290 295 300 Leu Glu Leu Ser His Asn Gln Ile Glu Glu Leu Pro Ser Leu His Arg 305 310 315 320 Cys Gln Lys Leu Glu Glu Ile Gly Leu Gln His Asn Arg Ile Trp Glu 325 330 335 Ile Gly Ala Asp Thr Phe Ser Gln Leu Ser Ser Leu Gln Ala Leu Asp 340 345 350 Leu Ser Trp Asn Ala Ile Arg Ser Ile His Pro Glu Ala Phe Ser Thr 355 360 365 Leu His Ser Leu Val Lys Leu Asp Leu Thr Asp Asn Gln Leu Thr Thr 370 375 380 Leu Pro Leu Ala Gly Leu Gly Gly Leu Met His Leu Lys Leu Lys Gly 385 390 395 400 Asn Leu Ala Leu Ser Gln Ala Phe Ser Lys Asp Ser Phe Pro Lys Leu 405 410 415 Arg Ile Leu Glu Val Pro Tyr Ala Tyr Gln Cys Cys Pro Tyr Gly Met 420 425 430 Cys Ala Ser Phe Phe Lys Ala Ser Gly Gln Trp Glu Ala Glu Asp Leu 435 440 445 His Leu Asp Asp Glu Glu Ser Ser Lys Arg Pro Leu Gly Leu Leu Ala 450 455 460 Arg Gln Ala Glu Asn His Tyr Asp Gln Asp Leu Asp Glu Leu Gln Leu 465 470 475 480 Glu Met Glu Asp Ser Lys Pro His Pro Ser Val Gln Cys Ser Pro Thr 485 490 495 Pro Gly Pro Phe Lys Pro Cys Glu Tyr Leu Phe Glu Ser Trp Gly Ile 500 505 510 Arg Leu Ala Val Trp Ala Ile Val Leu Leu Ser Val Leu Cys Asn Gly 515 520 525 Leu Val Leu Leu Thr Val Phe Ala Gly Gly Pro Val Pro Leu Pro Pro 530 535 540 Val Lys Phe Val Val Gly Ala Ile Ala Gly Ala Asn Thr Leu Thr Gly 545 550 555 560 Ile Ser Cys Gly Leu Leu Ala Ser Val Asp Ala Leu Thr Phe Gly Gln 565 570 575 Phe Ser Glu Tyr Gly Ala Arg Trp Glu Thr Gly Leu Gly Cys Arg Ala 580 585 590 Thr Gly Phe Leu Ala Val Leu Gly Ser Glu Ala Ser Val Leu Leu Leu 595 600 605 Thr Leu Ala Ala Val Gln Cys Ser Val Ser Val Ser Cys Val Arg Ala 610 615 620 Tyr Gly Lys Ser Pro Ser Leu Gly Ser Val Arg Ala Gly Val Leu Gly 625 630 635 640 Cys Leu Ala Leu Ala Gly Leu Ala Ala Ala Leu Pro Leu Ala Ser Val 645 650 655 Gly Glu Tyr Gly Ala Ser Pro Leu Cys Leu Pro Tyr Ala Pro Pro Glu 660 665 670 Gly Gln Pro Ala Ala Leu Gly Phe Thr Val Ala Leu Val Met Met Asn 675 680 685 Ser Phe Cys Phe Leu Val Val Ala Gly Ala Tyr Ile Lys Leu Tyr Cys 690 695 700 Asp Leu Pro Arg Gly Asp Phe Glu Ala Val Trp Asp Cys Ala Met Val 705 710 715 720 Arg His Val Ala Trp Leu Ile Phe Ala Asp Gly Leu Leu Tyr Cys Pro 725 730 735 Val Ala Phe Leu Ser Phe Ala Ser Met Leu Gly Leu Phe Pro Val Thr 740 745 750 Pro Glu Ala Val Lys Ser Val Leu Leu Val Val Leu Pro Val Pro Ala 755 760 765 Cys Leu Asn Pro Leu Leu Tyr Leu Leu Phe Asn Pro His Phe Arg Asp 770 775 780 Asp Leu Arg Arg Leu Arg Pro Arg Ala Gly Asp Ser Gly Pro Leu Ala 785 790 795 800 Tyr Ala Ala Ala Gly Glu Leu Glu Lys Ser Ser Cys Asp Ser Thr Gln 805 810 815 Ala Leu Val Ala Phe Ser Asp Val Asp Leu Ile Leu Glu Ala Ser Glu 820 825 830 Ala Gly Arg Pro Pro Gly Leu Glu Thr Tyr Gly Phe Pro Ser Val Thr 835 840 845 Leu Ile Ser Cys Gln Gln Pro Gly Ala Pro Arg Leu Glu Gly Ser His 850 855 860 Cys Val Glu Pro Glu Gly Asn His Phe Gly Asn Pro Gln Pro Ser Met 865 870 875 880 Asp Gly Glu Leu Leu Leu Arg Ala Glu Gly Ser Thr Pro Ala Gly Gly 885 890 895 Gly Leu Ser Gly Gly Gly Gly Phe Gln Pro Ser Gly Leu Ala Phe Ala 900 905 910 Ser His Val 915 6 823 PRT Human 6 Met Arg Leu Glu Gly Glu Gly Arg Ser Ala Arg Ala Gly Gln Asn Leu 1 5 10 15 Ser Arg Ala Gly Ser Ala Arg Arg Gly Ala Pro Arg Asp Leu Ser Met 20 25 30 Asn Asn Leu Thr Glu Leu Gln Pro Gly Leu Phe His His Leu Arg Phe 35 40 45 Leu Glu Glu Leu Arg Leu Ser Gly Asn His Leu Ser His Ile Pro Gly 50 55 60 Gln Ala Phe Ser Gly Leu Tyr Ser Leu Lys Ile Leu Met Leu Gln Asn 65 70 75 80 Asn Gln Leu Gly Gly Ile Pro Ala Glu Ala Leu Trp Glu Leu Pro Ser 85 90 95 Leu Gln Ser Leu Asp Leu Asn Tyr Asn Lys Leu Gln Glu Phe Pro Val 100 105 110 Ala Ile Arg Thr Leu Gly Arg Leu Gln Glu Leu Gly Phe His Asn Asn 115 120 125 Asn Ile Lys Ala Ile Pro Glu Lys Ala Phe Met Gly Asn Pro Leu Leu 130 135 140 Gln Thr Ile His Phe Tyr Asp Asn Pro Ile Gln Phe Val Gly Arg Ser 145 150 155 160 Ala Phe Gln Tyr Leu Pro Lys Leu His Thr Leu Ser Leu Asn Gly Ala 165 170 175 Met Asp Ile Gln Glu Phe Pro Asp Leu Lys Gly Thr Thr Ser Leu Glu 180 185 190 Ile Leu Thr Leu Thr Arg Ala Gly Ile Arg Leu Leu Pro Ser Gly Met 195 200 205 Cys Gln Gln Leu Pro Arg Leu Arg Val Leu Glu Leu Ser His Asn Gln 210 215 220 Ile Glu Glu Leu Pro Ser Leu His Arg Cys Gln Lys Leu Glu Glu Ile 225 230 235 240 Gly Leu Gln His Asn Arg Ile Trp Glu Ile Gly Ala Asp Thr Phe Ser 245 250 255 Gln Leu Ser Ser Leu Gln Ala Leu Asp Leu Ser Trp Asn Ala Ile Arg 260 265 270 Ser Ile His Pro Glu Ala Phe Ser Thr Leu His Ser Leu Val Lys Leu 275 280 285 Asp Leu Thr Asp Asn Gln Leu Thr Thr Leu Pro Leu Ala Gly Leu Gly 290 295 300 Gly Leu Met His Leu Lys Leu Lys Gly Asn Leu Ala Leu Ser Gln Ala 305 310 315 320 Phe Ser Lys Asp Ser Phe Pro Lys Leu Arg Ile Leu Glu Val Pro Tyr 325 330 335 Ala Tyr Gln Cys Cys Pro Tyr Gly Met Cys Ala Ser Phe Phe Lys Ala 340 345 350 Ser Gly Gln Trp Glu Ala Glu Asp Leu His Leu Asp Asp Glu Glu Ser 355 360 365 Ser Lys Arg Pro Leu Gly Leu Leu Ala Arg Gln Ala Glu Asn His Tyr 370 375 380 Asp Gln Asp Leu Asp Glu Leu Gln Leu Glu Met Glu Asp Ser Lys Pro 385 390 395 400 His Pro Ser Val Gln Cys Ser Pro Thr Pro Gly Pro Phe Lys Pro Cys 405 410 415 Glu Tyr Leu Phe Glu Ser Trp Gly Ile Arg Leu Ala Val Trp Ala Ile 420 425 430 Val Leu Leu Ser Val Leu Cys Asn Gly Leu Val Leu Leu Thr Val Phe 435 440 445 Ala Gly Gly Pro Val Pro Leu Pro Pro Val Lys Phe Val Val Gly Ala 450 455 460 Ile Ala Gly Ala Asn Thr Leu Thr Gly Ile Ser Cys Gly Leu Leu Ala 465 470 475 480 Ser Val Asp Ala Leu Thr Phe Gly Gln Phe Ser Glu Tyr Gly Ala Arg 485 490 495 Trp Glu Thr Gly Leu Gly Cys Arg Ala Thr Gly Phe Leu Ala Val Leu 500 505 510 Gly Ser Glu Ala Ser Val Leu Leu Leu Thr Leu Ala Ala Val Gln Cys 515 520 525 Ser Val Ser Val Ser Cys Val Arg Ala Tyr Gly Lys Ser Pro Ser Leu 530 535 540 Gly Ser Val Arg Ala Gly Val Leu Gly Cys Leu Ala Leu Ala Gly Leu 545 550 555 560 Ala Ala Ala Leu Pro Leu Ala Ser Val Gly Glu Tyr Gly Ala Ser Pro 565 570 575 Leu Cys Leu Pro Tyr Ala Pro Pro Glu Gly Gln Pro Ala Ala Leu Gly 580 585 590 Phe Thr Val Ala Leu Val Met Met Asn Ser Phe Cys Phe Leu Val Val 595 600 605 Ala Gly Ala Tyr Ile Lys Leu Tyr Cys Asp Leu Pro Arg Gly Asp Phe 610 615 620 Glu Ala Val Trp Asp Cys Ala Met Val Arg His Val Ala Trp Leu Ile 625 630 635 640 Phe Ala Asp Gly Leu Leu Tyr Cys Pro Val Ala Phe Leu Ser Phe Ala 645 650 655 Ser Met Leu Gly Leu Phe Pro Val Thr Pro Glu Ala Val Lys Ser Val 660 665 670 Leu Leu Val Val Leu Pro Leu Pro Ala Cys Leu Asn Pro Leu Leu Tyr 675 680 685 Leu Leu Phe Asn Pro His Phe Arg Asp Asp Leu Arg Arg Leu Arg Pro 690 695 700 Arg Ala Gly Asp Ser Gly Pro Leu Ala Tyr Ala Ala Ala Gly Glu Leu 705 710 715 720 Glu Lys Ser Ser Cys Asp Ser Thr Gln Ala Leu Val Ala Phe Ser Asp 725 730 735 Val Asp Leu Ile Leu Glu Ala Ser Glu Ala Gly Arg Pro Pro Gly Leu 740 745 750 Glu Thr Tyr Gly Phe Pro Ser Val Thr Leu Ile Ser Cys Gln Gln Pro 755 760 765 Gly Ala Pro Arg Leu Glu Gly Ser His Cys Val Glu Pro Glu Gly Asn 770 775 780 His Phe Gly Asn Pro Gln Pro Ser Met Asp Gly Glu Leu Leu Leu Arg 785 790 795 800 Ala Glu Gly Ser Thr Pro Ala Gly Gly Gly Leu Ser Gly Gly Gly Gly 805 810 815 Phe Gln Pro Ser Gly Leu Ala 820 7 794 PRT Human 7 Leu Ser Met Asn Asn Leu Thr Glu Leu Gln Pro Gly Leu Phe His His 1 5 10 15 Leu Arg Phe Leu Glu Glu Leu Arg Leu Ser Gly Asn His Leu Ser His 20 25 30 Ile Pro Gly Gln Ala Phe Ser Gly Leu Tyr Ser Leu Lys Ile Leu Met 35 40 45 Leu Gln Asn Asn Gln Leu Gly Gly Ile Pro Ala Glu Ala Leu Trp Glu 50 55 60 Leu Pro Ser Leu Gln Ser Leu Asp Leu Asn Tyr Asn Lys Leu Gln Glu 65 70 75 80 Phe Pro Val Ala Ile Arg Thr Leu Gly Arg Leu Gln Glu Leu Gly Phe 85 90 95 His Asn Asn Asn Ile Lys Ala Ile Pro Glu Lys Ala Phe Met Gly Asn 100 105 110 Pro Leu Leu Gln Thr Ile His Phe Tyr Asp Asn Pro Ile Gln Phe Val 115 120 125 Gly Arg Ser Ala Phe Gln Tyr Leu Pro Lys Leu His Thr Leu Ser Leu 130 135 140 Asn Gly Ala Met Asp Ile Gln Glu Phe Pro Asp Leu Lys Gly Thr Thr 145 150 155 160 Ser Leu Glu Ile Leu Thr Leu Thr Arg Ala Gly Ile Arg Leu Leu Pro 165 170 175 Ser Gly Met Cys Gln Gln Leu Pro Arg Leu Arg Val Leu Glu Leu Ser 180 185 190 His Asn Gln Ile Glu Glu Leu Pro Ser Leu His Arg Cys Gln Lys Leu 195 200 205 Glu Glu Ile Gly Leu Gln His Asn Arg Ile Trp Glu Ile Gly Ala Asp 210 215 220 Thr Phe Ser Gln Leu Ser Ser Leu Gln Ala Leu Asp Leu Ser Trp Asn 225 230 235 240 Ala Ile Arg Ser Ile His Pro Glu Ala Phe Ser Thr Leu His Ser Leu 245 250 255 Val Lys Leu Asp Leu Thr Asp Asn Gln Leu Thr Thr Leu Pro Leu Ala 260 265 270 Gly Leu Gly Gly Leu Met His Leu Lys Leu Lys Gly Asn Leu Ala Leu 275 280 285 Ser Gln Ala Phe Ser Lys Asp Ser Phe Pro Lys Leu Arg Ile Leu Glu 290 295 300 Val Pro Tyr Ala Tyr Gln Cys Cys Pro Tyr Gly Met Cys Ala Ser Phe 305 310 315 320 Phe Lys Ala Ser Gly Gln Trp Glu Ala Glu Asp Leu His Leu Asp Asp 325 330 335 Glu Glu Ser Ser Lys Arg Pro Leu Gly Leu Leu Ala Arg Gln Ala Glu 340 345 350 Asn His Tyr Asp Gln Asp Leu Asp Glu Leu Gln Leu Glu Met Glu Asp 355 360 365 Ser Lys Pro His Pro Ser Val Gln Cys Ser Pro Thr Pro Gly Pro Phe 370 375 380 Lys Pro Cys Glu Tyr Leu Phe Glu Ser Trp Gly Ile Arg Leu Ala Val 385 390 395 400 Trp Ala Ile Val Leu Leu Ser Val Leu Cys Asn Gly Leu Val Leu Leu 405 410 415 Thr Val Phe Ala Gly Gly Pro Val Pro Leu Pro Pro Val Lys Phe Val 420 425 430 Val Gly Ala Ile Ala Gly Ala Asn Thr Leu Thr Gly Ile Ser Cys Gly 435 440 445 Leu Leu Ala Ser Val Asp Ala Leu Thr Phe Gly Gln Phe Ser Glu Tyr 450 455 460 Gly Ala Arg Trp Glu Thr Gly Leu Gly Cys Arg Ala Thr Gly Phe Leu 465 470 475 480 Ala Val Leu Gly Ser Glu Ala Ser Val Leu Leu Leu Thr Leu Ala Ala 485 490 495 Val Gln Cys Ser Val Ser Val Ser Cys Val Arg Ala Tyr Gly Lys Ser 500 505 510 Pro Ser Leu Gly Ser Val Arg Ala Gly Val Leu Gly Cys Leu Ala Leu 515 520 525 Ala Gly Leu Ala Ala Ala Leu Pro Leu Ala Ser Val Gly Glu Tyr Gly 530 535 540 Ala Ser Pro Leu Cys Leu Pro Tyr Ala Pro Pro Glu Gly Gln Pro Ala 545 550 555 560 Ala Leu Gly Phe Thr Val Ala Leu Val Met Met Asn Ser Phe Cys Phe 565 570 575 Leu Val Val Ala Gly Ala Tyr Ile Lys Leu Tyr Cys Asp Leu Pro Arg 580 585 590 Gly Asp Phe Glu Ala Val Trp Asp Cys Ala Met Val Arg His Val Ala 595 600 605 Trp Leu Ile Phe Ala Asp Gly Leu Leu Tyr Cys Pro Val Ala Phe Leu 610 615 620 Ser Phe Ala Ser Met Leu Gly Leu Phe Pro Val Thr Pro Glu Ala Val 625 630 635 640 Lys Ser Val Leu Leu Val Val Leu Pro Leu Pro Ala Cys Leu Asn Pro 645 650 655 Leu Leu Tyr Leu Leu Phe Asn Pro His Phe Arg Asp Asp Leu Arg Arg 660 665 670 Leu Arg Pro Arg Ala Gly Asp Ser Gly Pro Leu Ala Tyr Ala Ala Ala 675 680 685 Gly Glu Leu Glu Lys Ser Ser Cys Asp Ser Thr Gln Ala Leu Val Ala 690 695 700 Phe Ser Asp Val Asp Leu Ile Leu Glu Ala Ser Glu Ala Gly Arg Pro 705 710 715 720 Pro Gly Leu Glu Thr Tyr Gly Phe Pro Ser Val Thr Leu Ile Ser Cys 725 730 735 Gln Gln Pro Gly Ala Pro Arg Leu Glu Gly Ser His Cys Val Glu Pro 740 745 750 Glu Gly Asn His Phe Gly Asn Pro Gln Pro Ser Met Asp Gly Glu Leu 755 760 765 Leu Leu Arg Ala Glu Gly Ser Thr Pro Ala Gly Gly Gly Leu Ser Gly 770 775 780 Gly Gly Gly Phe Gln Pro Ser Gly Leu Ala 785 790 

That which is claimed is:
 1. An isolated peptide consisting of an amino acid sequence selected from the group consisting of: (a) an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5; (b) an amino acid sequence of an allelic variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of SEQ ID NOS:1 (transcript), 3 (genomic), and 4 (transcript); (c) an amino acid sequence of an ortholog of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of SEQ ID NOS:1 (transcript), 3 (genomic), and 4 (transcript); and (d) a fragment of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said fragment comprises at least 10 contiguous amino acids.
 2. An isolated peptide comprising an amino acid sequence selected from the group consisting of: (a) an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5; (b) an amino acid sequence of an allelic variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of SEQ ID NOS:1 (transcript), 3 (genomic), and 4 (transcript); (c) an amino acid sequence of an ortholog of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of SEQ ID NOS:1 (transcript), 3 (genomic), and 4 (transcript); and (d) a fragment of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said fragment comprises at least 10 contiguous amino acids.
 3. An isolated antibody that selectively binds to a peptide of claim
 2. 4. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5; (b) a nucleotide sequence that encodes an allelic variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of SEQ ID NOS:1 (transcript), 3 (genomic), and 4 (transcript); (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of SEQ ID NOS:1 (transcript), 3 (genomic), and 4 (transcript); (d) a nucleotide sequence that encodes a fragment of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d).
 5. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5; (b) a nucleotide sequence that encodes an allelic variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of SEQ ID NOS:1 (transcript), 3 (genomic), and 4 (transcript); (c) a nucleotide sequence that encodes an ortholog of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule selected from the group consisting of SEQ ID NOS:1 (transcript), 3 (genomic), and 4 (transcript); (d) a nucleotide sequence that encodes a fragment of an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and 5, wherein said fragment comprises at least 10 contiguous amino acids; and (e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d).
 6. A gene chip comprising a nucleic acid molecule of claim
 5. 7. A transgenic non-human animal comprising a nucleic acid molecule of claim
 5. 8. A nucleic acid vector comprising a nucleic acid molecule of claim
 5. 9. A host cell containing the vector of claim
 8. 10. A method for producing any of the peptides of claim 1 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.
 11. A method for producing any of the peptides of claim 2 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.
 12. A method for detecting the presence of any of the peptides of claim 2 in a sample, said method comprising contacting said sample with a detection agent that specifically allows detection of the presence of the peptide in the sample and then detecting the presence of the peptide.
 13. A method for detecting the presence of a nucleic acid molecule of claim 5 in a sample, said method comprising contacting the sample with an oligonucleotide that hybridizes to said nucleic acid molecule under stringent conditions and determining whether the oligonucleotide binds to said nucleic acid molecule in the sample.
 14. A method for identifying a modulator of a peptide of claim 2, said method comprising contacting said peptide with an agent and determining if said agent has modulated the function or activity of said peptide.
 15. The method of claim 14, wherein said agent is administered to a host cell comprising an expression vector that expresses said peptide.
 16. A method for identifying an agent that binds to any of the peptides of claim 2, said method comprising contacting the peptide with an agent and assaying the contacted mixture to determine whether a complex is formed with the agent bound to the peptide.
 17. A pharmaceutical composition comprising an agent identified by the method of claim 16 and a pharmaceutically acceptable carrier therefor.
 18. A method for treating a disease or condition mediated by a human proteases, said method comprising administering to a patient a pharmaceutically effective amount of an agent identified by the method of claim
 16. 19. A method for identifying a modulator of the expression of a peptide of claim 2, said method comprising contacting a cell expressing said peptide with an agent, and determining if said agent has modulated the expression of said peptide.
 20. An isolated human protease peptide having an amino acid sequence that shares at least 70% homology with an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and
 5. 21. A peptide according to claim 20 that shares at least 90 percent homology with an amino acid sequence selected from the group consisting of SEQ ID NOS:2 and
 5. 22. An isolated nucleic acid molecule encoding a human protease peptide, said nucleic acid molecule sharing at least 80 percent homology with a nucleic acid molecule selected from the group consisting of SEQ ID NOS:1 (transcript), 3 (genomic), and 4 (transcript).
 23. A nucleic acid molecule according to claim 22 that shares at least 90 percent homology with a nucleic acid molecule selected from the group consisting of SEQ ID NOS:1 (transcript), 3 (genomic), and 4 (transcript). 